Delta-8 desaturases and their use in making polyunsaturated fatty acids

ABSTRACT

The present invention relates to Δ8 desaturase genes, which have the ability to convert eicosadienoic acid (EDA; 20:2 ω-6) to dihomo-γ-linolenic acid (DGLA; 20:3 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3) to eicosatetraenoic acid (ETA; (20:3 ω-3). Isolated nucleic acid fragments and recombinant DNA constructs comprising such fragments encoding Δ8 desaturases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these Δ8 desaturases in oleaginous yeast are disclosed.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/910,831, filed Apr. 10, 2007, which is incorporatedherein in its entirety by reference.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the identification of polynucleotide sequencesencoding Δ8 fatty acid desaturases and the use of these desaturases inmaking long-chain polyunsaturated fatty acids (PUFAs).

BACKGROUND OF THE INVENTION

Today, a variety of different hosts including plants, algae, fungi,stramenopiles and yeast are being investigated as means for commercialPUFA production. Genetic engineering has demonstrated that the naturalabilities of some hosts (even those natively limited to linoleic acid(LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3) fatty acidproduction) can be substantially altered to result in high-levelproduction of various long-chain ω-3/ω-6 PUFAs. Accordingly, productionof arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) may require expression ofa Δ8 desaturase.

The Δ8 desaturase enzymes identified thus far have the ability toconvert both eicosadienoic acid (EDA; 20:2 ω-6) to dihomo-γ-linolenicacid (DGLA; 20:3 ω-6) and eicosatrienoic acid (ETrA; 20:3 ω-3) toeicosatetraenoic acid (ETA; 20:4 ω-3) (wherein ARA are EPA aresubsequently synthesized from DGLA and ETA, respectively, followingreaction with a Δ5 desaturase, while DHA synthesis requires subsequentexpression of an additional C_(20/22) elongase and a Δ4 desaturase).

Based on the role Δ8 desaturase enzymes play in the synthesis of e.g.,ARA, EPA and DHA, considerable effort has been made to identify andcharacterize these enzymes from various sources. Initial efforts havefocused on the isolation and characterization of Δ8 desaturases fromEuglena gracilis; and, several sequence variations within the Euglenagracilis Δ8 desaturase have been reported (see, e.g., Wallis et al.,Arch. Biochem. and Biophys., 365(2):307-316 (1999); PCT Publication No.WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO2004/057001). Additionally, commonly owned, co-pending U.S. applicationSer. No. 11/166,003 and U.S. Pat. No. 7,256,033 disclose amino acid andnucleic acid sequences for a Euglena gracilis Δ8 desaturase. In otherwork commonly owned, co-pending applications U.S. patent applicationsSer. No. 11/635258 and Ser. No. 11/951697 describe a syntheticallyengineered mutant Δ8 desaturase, derived from Euglena gracilis.

U.S. Publication No. 2005/0273885 discloses amino acid and nucleic acidsequences for a Δ8 desaturase enzyme from Pavlova salina and commonlyowned and co-pending application U.S. patent application Ser. No.11/737772 discloses amino acid and nucleic acid sequences for a Δ8desaturase enzyme from Pavlova lutheri (CCMP459), whereas U.S. patentapplication Ser. No. 11/876115 discloses amino acid and nucleic acidsequences for Δ8 desaturase enzymes from Tetruetreptia pomquetensisCCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf _(—) gymnasticaCCMP1594. Sayanova et al. (FEBS Lett., 580:1946-1952 (2006)) describethe isolation and characterization of a cDNA from the free living soilamoeba Acanthamoeba castellanii that, when expressed in Arabidopsis,encodes a polypeptide having C₂₀ Δ8 desaturase activity.

Despite the disclosures cited above, there is a need for additionalgenes encoding polypeptides having Δ8 desaturase activity as it is onlythrough genetic variation that a wide variety of host cells may beoptimized for PUFA production. Applicants address the stated need hereinby reporting the isolation of genes encoding Δ8 fatty acid desaturasesfrom Euglena anabaena.

SUMMARY OF THE INVENTION

The present invention relates to new genetic constructs encodingpolypeptides having Δ8 desaturase activity, and their use in algae,bacteria, yeast, euglenoids, stramenopiles and fungi for the productionof PUFAs. Accordingly the invention provides, a microbial host cellcomprising an isolated polynucleotide comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having Δ8        desaturase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence selected from the group        consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ        ID NO:24;    -   (b) a nucleotide sequence encoding a polypeptide having Δ8        desaturase activity, wherein the nucleotide sequence has at        least 80% sequence identity, based on the BLASTN method of        alignment, when compared to a nucleotide sequence selected from        the group consisting of: SEQ ID NO:17, SEQ ID NO:18, SEQ ID        NO:19, SEQ ID NO:20 and SEQ ID NO:39;    -   (c) a nucleotide sequence encoding a polypeptide having Δ8        desaturase activity, wherein the nucleotide sequence hybridizes        under stringent conditions to a nucleotide sequence selected        from the group consisting of: SEQ ID NO:17, SEQ ID NO:18, SEQ ID        NO:19, SEQ ID NO:20 and SEQ ID NO:39; or    -   (d) a complement of the nucleotide sequence of (a), (b) or (c),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary.

In another embodiment the invention provides a method for the productionof dihomo-γ-linoleic acid comprising:

-   -   a) providing a microbial host cell comprising:        -   (i) a recombinant nucleotide molecule encoding a Δ8            desaturase polypeptide having at least 80% amino acid            identity, based on the Clustal V method of alignment, when            compared to a polypeptide having an amino acid sequence            selected from the group consisting of: SEQ ID NO:21, SEQ ID            NO:22, SEQ ID NO:23 and SEQ ID NO:24; and,        -   (ii) a source of eicosadienoic acid;    -   b) growing the microbial host cell of step (a) under conditions        wherein the nucleic acid fragment encoding the Δ8 desaturase        polypeptide is expressed and the eicosadienoic acid is converted        to dihomo-γ-linoleic acid; and,        optionally recovering the dihomo-γ-linoleic acid of step (b).

Similarly the invention provides a method for the production ofeicosatetraenoic acid comprising:

-   -   a) providing a microbial host cell comprising:        -   (i) a recombinant nucleotide molecule encoding a Δ5            desaturase polypeptide having at least 80% amino acid            identity, based on the Clustal V method of alignment, when            compared to a polypeptide having an amino acid sequence            selected from the group consisting of: SEQ ID NO:21, SEQ ID            NO:22, SEQ ID NO:23 and SEQ ID NO:24; and,        -   (ii) a source of eicosatrienoic acid;    -   b) growing the microbial host cell of step (a) under conditions        wherein the nucleic acid fragment encoding the Δ8 desaturase        polypeptide is expressed and the eicosatrienoic acid is        converted to eicosatetraenoic acid; and,    -   c) optionally recovering the eicosatetraenoic acid of step (b).

In another embodiment the invention provides an isolated nucleic acidmolecule which encodes a Δ8 desaturase as set forth in SEQ ID NO:39wherein at least 208 codons are codon-optimized for expression inYarrowia sp.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

FIG. 1 is a representative ω-3 and ω-6 fatty acid biosynthetic pathwayproviding for the conversion of myristic acid through variousintermediates to DHA.

FIG. 2A diagrams the development of Yarrowia lipolytica strain Y4001U,producing about 17% EDA in the total lipid fraction. FIG. 2B providesthe plasmid map for pZKLeuN-29E3, while FIG. 2C provides the plasmid mapfor pY116.

FIG. 3 shows a chromatogram of the lipid profile of an Euglena anabaenacell extract as described in Example 1.

FIGS. 4A, 4B and 4C show a Clustal V alignment of the Δ8 desaturasesequences for EaD8Des1 (SEQ ID NO:21), EaD8Des2 (SEQ ID NO:22), EaD8Des3(SEQ ID NO:23) and EaD8Des4 (SEQ ID NO:24), and a functional variantEuglena gracilis Δ8 desaturase amino acid sequence (EgD8; SEQ ID NO:25;described as Eg5 in PCT Application No. WO 2006/012325).

FIG. 5 provides plasmid maps for the following: (A) pY115 (SEQ IDNO:34); and, (B) pY175 (SEQ ID NO:35).

FIG. 6 provides the fatty acid profiles for Yarrowia lipolyticaexpressing pY175, pY176, pY177 and pY178 (see Example 5).

FIGS. 7A and 7B show a comparison of the nucleotide sequences ofEaD8Des3 (SEQ ID NO:19) and EaD8S (SEQ ID NO:39).

FIG. 8 provides plasmid maps for the following: (A) pEaD8S (SEQ IDNO:41); and, (B) pZUFmEaD8S (SEQ ID NO:51).

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1, 2, 10, 11, 13-25, and 28-51 are ORFs encoding genes orproteins (or portions thereof), or plasmids, as identified in Table 1.

TABLE 1 Summary Of Nucleic Acid And Protein SEQ ID Numbers ProteinNucleic acid SEQ Description and Abbreviation SEQ ID NO. ID NO. Euglenaanabaena Δ8 desaturase partial  1 — sequence  (604 bp) Euglena gracilisΔ8 desaturase CDS (“Eg5”)  2 — (1263 bp) Plasmid pHD23-1 10 — (4116 bp)Euglena gracilis Δ8 desaturase (NCBI — 11 Accession No. AAD45877) (419AA) Plasmid pLF118-1 13 — (4363 bp) Plasmid pLF118-2 14 — (4307 bp)Plasmid pLF118-3 15 — (4307 bp) Plasmid pLF118-4 16 — (4297 bp) Euglenaanabaena Δ8 desaturase 1 coding 17 21 sequence (“EaD8Des1”) (1260 bp)(420 AA) Euglena anabaena Δ8 desaturase 2 coding 18 22 sequence(“EaD8Des2”) (1260 bp) (420 AA) Euglena anabaena Δ8 desaturase 3 coding19 23 sequence (“EaD8Des3”) (1260 bp) (420 AA) Euglena anabaena Δ8desaturase 4 coding 20 24 sequence (“EaD8Des4”) (1260 bp) (420 AA)Euglena gracilis Δ8 desaturase (“EgD8”) — 25 (U.S. Pat. No. 7,256,033)(421 AA) Plasmid pLF120-1 28 — (4794 bp) Plasmid pLF120-2 29 — (4794 bp)Plasmid pLF120-3 30 — (4794 bp) Plasmid pLF120-4 31 — (4794 bp) plasmidpDMW263 32 — (9472 bp) plasmid pDMW237 33 — (7879 bp) plasmid pY115 34 —(7783 bp) plasmid pY175 35 — (8254 bp) plasmid pY176 36 — (8254 bp)plasmid pY177 37 — (8254 bp) plasmid pY178 38 — (8254 bp) Synthetic Δ8desaturase, derived from 39 40 Euglena anabaena, codon-optimized for(1260 bp) (420 AA) expression in Yarrowia lipolytica (“EaD8S”) PlasmidpEaD8S 41 — (3983 bp) Plasmid pZKLeuN-29E3 42 — (14,688 bp)   Fusariummoniliforme Δ12 desaturase 43 44 (“FmD12”) (1434 bp) (477 AA) SyntheticΔ9 elongase derived from Euglena 45 46 gracilis, codon-optimized forexpression in  (777 bp) (258 AA) Yarrowia lipolytica (“EgD9eS”)Escherichia coli LoxP recombination site, 47 — recognized by a Crerecombinase enzyme  (34 bp) Synthetic C_(16/18) elongase derived from 4849 Mortierella alpina ELO3, codon-optimized  (828 bp) (275 AA) forexpression in Yarrowia lipolytica (“ME3S”) Plasmid pY116 50 — (8739 bp)Plasmid pZUFmEaD8S 51 — (8255 bp)

SEQ ID NO:3 is the nucleotide sequence of the vector-specific primerpDonor222Eg5-1.

SEQ ID NOs:4-7 correspond to degenerate primers D8DEG3-1, D8DEG3-2,D8DEG3-3 and D8DEG3-4, respectively, used to amplify a portion of the Δ8desaturase genes from Euglena anabaena UTEX 373.

SEQ ID NOs:8 and 9 correspond to the T7 primer and primer M13-28Rev,respectively, used for sequencing a partial putative Δ8 desaturase cDNAfragment.

SEQ ID NO:12 is the nucleotide sequence of primer EaD8seq-1, used forfull insert sequencing of eug1c Δ8 desaturase clones.

SEQ ID NOs:26 and 27 correspond to primers EaD8-5 and EaD8-3,respectively, used to amplify the EaD8Des1, EaD8Des2, EaD8Des3 andEaD8Des4 coding sequences.

DETAILED DESCRIPTION OF THE INVENTION

New Euglena anabaena Δ8 desaturase enzymes and genes encoding the samethat may be used for the manipulation of biochemical pathways for theproduction of healthful PUFAs are disclosed herein. PUFAs, orderivatives thereof, are used as dietary substitutes, or supplements,particularly infant formulas, for patients undergoing intravenousfeeding or for preventing or treating malnutrition. Alternatively, thepurified PUFAs (or derivatives thereof) may be incorporated into cookingoils, fats or margarines formulated so that in normal use the recipientwould receive the desired amount for dietary supplementation. The PUFAsmay also be incorporated into infant formulas, nutritional supplementsor other food products and may find use as anti-inflammatory orcholesterol lowering agents. Optionally, the compositions may be usedfor pharmaceutical use (human or veterinary).

Definitions

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Triacylglycerols” are abbreviated TAGs.

The term “invention” or “present invention” as used herein is not meantto be limiting to any one specific embodiment of the invention butapplies generally to any and all embodiments of the invention asdescribed in the claims and specification.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

The term “fatty acids” refers to long-chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” (or “PUFAs”), and “omega-6 fatty acids” (ω-6 or n-6) versus“omega-3 fatty acids” (ω-3 or n-3) are provided in U.S. Pat. No.7,238,482.

Fatty acids are described herein by a simple notation system of “X:Y”,where X is the total number of carbon (C) atoms in the particular fattyacid and Y is the number of double bonds. The number following the fattyacid designation indicates the position of the double bond from thecarboxyl end of the fatty acid with the “c” affix for thecis-configuration of the double bond (e.g., palmitic acid (16:0),stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1,6c), LA (18:2, 9c,12c), GLA (18:3, 6c,9c,12c) and ALA (18:3,9c,12c,15c)). Unless otherwise specified, 18:1, 18:2 and 18:3 refer tooleic, LA and ALA fatty acids, respectively. If not specifically writtenas otherwise, double bonds are assumed to be of the cis configuration.For instance, the double bonds in 18:2 (9,12) would be assumed to be inthe cis configuration.

Nomenclature used to describe PUFAs in the present disclosure is shownbelow in Table 2. In the column titled “Shorthand Notation”, theomega-reference system is used to indicate the number of carbons, thenumber of double bonds and the position of the double bond closest tothe omega carbon, counting from the omega carbon (which is numbered 1for this purpose). The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids and their precursors, the abbreviationsthat will be used throughout the remainder of the specification, andeach compounds' chemical name.

TABLE 2 Nomenclature of Polyunsaturated Fatty Acids and PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic PA or hexadecanoic 16:0 PalmitatePalmitoleic — 9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic —cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6γ-Linolenic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDAcis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLAcis-8,11,14-eicosatrienoic 20:3 ω-6 linolenic Sciadonic SCIcis-5,11,14-eicosatrienoic 20:3b ω-6 Arachidonic ARA cis-5,8,11,14- 20:4ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 ω-3octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3octadecatetraenoic Eicosatrienoic ETrA or ERA cis-11,14,17- 20:3 ω-3eicosatrienoic Eicosatetraenoic ETA cis-8,11,14,17- 20:4 ω-3eicosatetraenoic Juniperonic JUP cis-5,11,14,17- 20:4b ω-3eicosatrienoic Docosatrienoic DRA cis-10,13,16- 22:3 ω-6 docosatrienoicDocosatetraenoic DTA cis-7,10,13,16- 22:4 ω-6 docosatetraenoicDocosapentaenoic DPAn-6 cis-4,7,10,13,16- 22:5 ω-6 docosapentaenoicEicosapentaenoic EPA cis-5,8,11,14,17- 20:5 ω-3 eicosapentaenoicDocosapentaenoic DPA cis-7,10,13,16,19- 22:5 ω-3 docosapentaenoicDocosahexaenoic DHA cis-4,7,10,13,16,19- 22:6 ω-3 docosahexaenoic

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

“Percent (%) PUFAs in the total lipid and oil fractions” refers to thepercent of PUFAs relative to the total fatty acids in those fractions.The term “total lipid fraction” or “lipid fraction” both refer to thesum of all lipids (i.e., neutral and polar) within an oleaginousorganism, thus including those lipids that are located in thephosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fractionand triacylglycerol (TAG or oil) fraction. However, the terms “lipid”and “oil” will be used interchangeably throughout the specification.

A metabolic pathway, or biosynthetic pathway, in a biochemical sense,can be regarded as a series of chemical reactions occurring within acell, catalyzed by enzymes, to achieve either the formation of ametabolic product to be used or stored by the cell, or the initiation ofanother metabolic pathway (then called a flux generating step). Many ofthese pathways are elaborate, and involve a step by step modification ofthe initial substance to shape it into a product having the exactchemical structure desired.

The term “PUFA biosynthetic pathway” refers to a metabolic process thatconverts oleic acid to ω-6 fatty acids such as LA, EDA, GLA, DGLA, ARA,DRA, DTA and DPAn-6 and ω-3 fatty acids such as ALA, STA, ETrA, ETA,EPA, DPA and DHA. This process is well described in the literature(e.g., see PCT Publication No. WO 2006/052870). Briefly, this processinvolves elongation of the carbon chain through the addition of carbonatoms and desaturation of the molecule through the addition of doublebonds, via a series of special desaturation and elongation enzymes(i.e., “PUFA biosynthetic pathway enzymes”) present in the endoplasmicreticulim membrane. More specifically, “PUFA biosynthetic pathwayenzyme” refers to any of the following enzymes (and genes which encodesaid enzymes) associated with the biosynthesis of a PUFA, including: aΔ9 elongase, a C_(14/16) elongase, a C_(16/18) elongase, a C_(18/20)elongase, a C_(20/22) elongase, a Δ4 desaturase, a Δ5 desaturase, a Δ6desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9desaturase and/or a Δ8 desaturase.

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode PUFA biosynthetic pathway enzymes. Arepresentative pathway is illustrated in FIG. 1, providing for theconversion of myristic acid through various intermediates to DHA, whichdemonstrates how both ω-3 and ω-6 fatty acids may be produced from acommon source. The pathway is naturally divided into two portions whereone portion will generate ω-3 fatty acids and the other portion, ω-6fatty acids.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all) of the genes in thepathway express active enzymes, resulting in in vivo catalysis orsubstrate conversion. It should be understood that “ω-3/ω-6 fatty acidbiosynthetic pathway” or “functional ω-3/ω-6 fatty acid biosyntheticpathway” does not imply that all the PUFA biosynthetic pathway enzymegenes are required, as a number of fatty acid products will only requirethe expression of a subset of the genes of this pathway.

The term “Δ6 desaturase/Δ6 elongase pathway” will refer to a PUFAbiosynthetic pathway that minimally includes at least one Δ6 desaturaseand at least one C_(18/20) elongase (also referred to as a Δ6 elongase),thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA,respectively, with GLA and/or STA as intermediate fatty acids. Withexpression of other desaturases and elongases, ARA, EPA, DPA and DHA mayalso be synthesized.

The term “Δ9 elongase/Δ8 desaturase pathway” will refer to a PUFAbiosynthetic pathway that minimally includes at least one Δ9 elongaseand at least one Δ8 desaturase, thereby enabling biosynthesis of DGLAand/or ETA from LA and ALA, respectively, with EDA and/or ETrA asintermediate fatty acids. With expression of other desaturases andelongases, ARA, EPA, DPA and DHA may also be synthesized.

The term “intermediate fatty acid” refers to any fatty acid produced ina fatty acid metabolic pathway that can be further converted to anintended product fatty acid in this pathway by the action of othermetabolic pathway enzymes. For instance, when EPA is produced using theΔ9 elongase/Δ8 desaturase pathway, EDA, ETrA, DGLA, ETA and ARA can beproduced and are considered “intermediate fatty acids” since these fattyacids can be further converted to EPA via action of other metabolicpathway enzymes.

The term “by-product fatty acid” refers to any fatty acid produced in afatty acid metabolic pathway that is not the intended fatty acid productof the pathway nor an “intermediate fatty acid” of the pathway. Forinstance, when EPA is produced using the Δ9 elongase/Δ8 desaturasepathway, sciadonic acid (SCI) and juniperonic acid (JUP) also can beproduced by the action of a Δ5 desaturase on either EDA or ETrA,respectively. They are considered to be “by-product fatty acids” sinceneither can be further converted to EPA by the action of other metabolicpathway enzymes.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are Δ8 desaturases that desaturate a fattyacid between the eighth and ninth carbon atom numbered from thecarboxyl-terminal end of the molecule and that can, for example,catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Other fattyacid desaturases include, for example: (1) Δ5 desaturases that catalyzethe conversion of DGLA to ARA and/or ETA to EPA; (2) Δ6 desaturases thatcatalyze the conversion of LA to GLA and/or ALA to STA; (3) Δ4desaturases that catalyze the conversion of DPA to DHA and/or DTA toDPAn-6; (4) Δ12 desaturases that catalyze the conversion of oleic acidto LA; (5) Δ15 desaturases that catalyze the conversion of LA to ALAand/or GLA to STA; (6) Δ17 desaturases that catalyze the conversion ofARA to EPA and/or DGLA to ETA; and, (7) Δ9 desaturases that catalyze theconversion of palmitic acid to palmitoleic acid (16:1) and/or stearicacid to oleic acid (18:1). In the art, Δ15 and Δ17 desaturases are alsooccasionally referred to as “omega-3 desaturases”, “w-3 desaturases”and/or “ω-3 desaturases”, based on their ability to convert ω-6 fattyacids into their ω-3 counterparts (e.g., conversion of LA into ALA andARA into EPA, respectively). In some embodiments, it may be mostdesirable to empirically determine the specificity of a particular fattyacid desaturase by transforming a suitable host with the gene for thefatty acid desaturase and determining its effect on the fatty acidprofile of the host.

The term “EaD8Des1” refers to a Δ8 desaturase enzyme (SEQ ID NO:21)isolated from Euglena anabaena, encoded by SEQ ID NO:17 herein. The term“EaD8Des2” refers to a Δ8 desaturase enzyme (SEQ ID NO:22) isolated fromE. anabaena, encoded by SEQ ID NO:18 herein. Likewise, the term“EaD8Des3” refers to a Δ8 desaturase enzyme (SEQ ID NO:23) isolated fromE. anabaena, encoded by SEQ ID NO:19 herein. The term “EaD8Des4” refersto a Δ8 desaturase enzyme (SEQ ID NO:24) isolated from E. anabaena,encoded by SEQ ID NO:20 herein. Similarly, the term “EaD8S” refers to asynthetic Δ8 desaturase derived from E. anabaena that is codon-optimizedfor expression in Yarrowia lipolytica (i.e., SEQ ID NOs:39 and 40).

The term “EgD8” refers to a Δ8 desaturase enzyme (encoded by thenucleotide sequence set forth as SEQ ID NO:2) isolated from Euglenagracilis. EgD8 is 100% identical and functionally equivalent to “Eg5”,as described in PCT Publication Nos. WO 2006/012325 and WO 2006/012326(i.e., SEQ ID NO:2 of U.S. Pat. No. 7,256,033).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., adesaturase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid that is 2 carbons longer than thefatty acid substrate that the elongase acts upon. This process ofelongation occurs in a multi-step mechanism in association with fattyacid synthase, as described in U.S. Patent Publication No. 2005/0132442.Examples of reactions catalyzed by elongase systems are the conversionof GLA to DGLA, STA to ETA, LA to EDA, ALA to ETrA, ARA to DTA and EPAto DPA.

In general, the substrate selectivity of elongases is somewhat broad butsegregated by both chain length and the degree of unsaturation. Forexample, a C_(14/16) elongase will utilize a C₁₄ substrate (e.g.,myristic acid), a C_(16/18) elongase will utilize a C₁₆ substrate (e.g.,palmitate), a C_(18/20) elongase (also known as a Δ6 elongase as theterms can be used interchangeably) will utilize a C₁₈ substrate (e.g.,GLA, STA) and a C_(20/22) elongase will utilize a C₂₀ substrate (e.g.,ARA, EPA). Similarily, a “Δ9 elongase” is able to catalyze theconversion of LA to EDA and/or ALA to ETrA. It is important to note thatsome elongases have broad specificity and thus a single enzyme may becapable of catalyzing several elongase reactions (e.g., thereby actingas both a C_(16/18) elongase and a C_(18/20) elongase). It may bedesirable to empirically determine the specificity of a fatty acidelongase by transforming a suitable host with the gene for the fattyacid elongase and determining its effect on the fatty acid profile ofthe host.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) Ed., Plenum, 1980). Within oleaginousmicroorganisms the cellular oil or TAG content generally follows asigmoid curve, wherein the concentration of lipid increases until itreaches a maximum at the late logarithmic or early stationary growthphase and then gradually decreases during the late stationary and deathphases (Yongmanitchai and Ward, Appl. Environ. Microbiol., 57:419-25(1991)). It is not uncommon for oleaginous microorganisms to accumulatein excess of about 25% of their dry cell weight as oil.

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. Examples of oleaginous yeast include, but areno means limited to, the following genera: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.Preferred for use in the present invention are oleaginous strains ofYarrowia lipolytica.

The term “Euglenophyceae” refers to a group of unicellular colorless orphotosynthetic flagellates (“euglenoids”) found living in freshwater,marine, soil and parasitic environments. The class is characterized bysolitary unicells, wherein most are free-swimming and have two flagella(one of which may be nonemergent) arising from an anterior invaginationknown as a reservoir. Photosynthetic euglenoids contain one to manychloroplasts, which vary from minute disks to expanded plates orribbons. Colorless euglenoids depend on osmotrophy or phagotrophy fornutrient assimilation. About 1000 species have been described andclassified into about 40 genera and 6 orders. Examples of Euglenophyceaeinclude, but are no means limited to, the following genera: Euglena,Eutreptiella and Tetruetreptia.

As used herein, “nucleic acid” means a polynucleotide and includessingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” or “nucleic acid fragment” are usedinterchangeably and refer to a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. A polynucleotide in the form of a polymer of DNA maybe comprised of one or more segments of cDNA, genomic DNA, syntheticDNA, or mixtures thereof. Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deosycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridlate, “T” for deosythymidylate,“R” for purines (A or G), “Y” for pyrimidiens (C or T), “K” for G or T,“H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize (under moderately stringent conditions, e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, theimportant factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth et al., Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); and, low stringency conditions canutilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (T_(m)). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120 or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the results by 100 to yield the percentage of sequenceidentity. Useful examples of percent sequence identities include, butare not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%,or any integer percentage from 50% to 100%. These identities can bedetermined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS, 5:151-153(1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992))and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments,the default values correspond to GAP PENALTY=10 and GAP LENGTHPENALTY=10. Default parameters for pairwise alignments and calculationof percent identity of protein sequences using the Clustal V method areKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4. After alignment of the sequences using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table in the same program.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the instant euglenoidpolypeptides as set forth in SEQ ID NOs:21, 22, 23 and 24. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. Accordingly, the genes can be tailored for optimal gene expressionbased on optimization of nucleotide sequence to reflect the codon biasof the host cell. The skilled artisan appreciates the likelihood ofsuccessful gene expression if codon usage is biased towards those codonsfavored by the host. Determination of preferred codons can be based on asurvey of genes derived from the host cell, where sequence informationis available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. A“codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

A promoter sequence may consist of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter.

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., Mol.Biotechnol., 3:225-236 (1995)).

The terms “3′ non-coding sequences”, “transcription terminator” and“termination sequences” refer to DNA sequences located downstream of acoding sequence. This includes polyadenylation recognition sequences andother sequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The 3′ region can influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065). The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. The term “recombinant” refers to an artificial combinationof two otherwise separated segments of sequence, e.g., by chemicalsynthesis or by the manipulation of isolated segments of nucleic acidsby genetic engineering techniques.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragments of the invention. Expression may also refer totranslation of mRNA into a protein (either precursor or mature).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes that are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA fragments.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing anexpression cassette(s) into a cell.

The term “expression cassette” refers to a fragment of DNA comprisingthe coding sequence of a selected gene and regulatory sequencespreceding (5′ non-coding sequences) and following (3′ non-codingsequences) the coding sequence that are required for expression of theselected gene product. Thus, an expression cassette is typicallycomposed of: (1) a promoter sequence; (2) a coding sequence (i.e., ORF);and, (3) a 3′ untranslated region (i.e., a terminator) that, ineukaryotes, usually contains a polyadenylation site. The expressioncassette(s) is usually included within a vector, to facilitate cloningand transformation. Different expression cassettes can be transformedinto different organisms including bacteria, yeast, plants and mammaliancells, as long as the correct regulatory sequences are used for eachhost.

A “recombinant DNA construct” (also referred to interchangeably hereinas a “expression construct” and “construct”) comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that will be used to transform host cells as is well known tothose skilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al., EMBO J., 4:2411-2418(1985); De Almeida et al., Mol. Gen. Genetics, 218:78-86 (1989)), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others.

The term “introduced” means providing a nucleic acid (e.g., expressioncassette) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct or expression cassette) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms or “recombinant” or“transformed” organisms.

As used herein, “transgenic” refers to a cell which comprises within itsgenome a heterologous polynucleotide. Preferably, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of anexpression cassette. Transgenic is used herein to include any cell orcell line, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989); bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984);and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken,N.J. (1987). Transformation methods are well known to those skilled inthe art and are described infra. An Overview: Microbial Biosynthesis OfFatty Acids And Triacylglycerols

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0) in oleaginous microorganisms, is described in detail inU.S. Pat. No. 7,238,482. Palmitate is the precursor of longer-chainsaturated and unsaturated fatty acid derivates, which are formed throughthe action of elongases and desaturases (FIG. 1).

TAGs (the primary storage unit for fatty acids) are formed by a seriesof reactions that involve: (1) the esterification of one molecule ofacyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; (2) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); (3) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and,(4) the addition of a third fatty acid by the action of anacyltransferase to form TAG. A wide spectrum of fatty acids can beincorporated into TAGs, including saturated and unsaturated fatty acidsand short-chain and long-chain fatty acids.

Biosynthesis of Omega Fatty Acids

The metabolic process wherein oleic acid is converted to ω-3/ω-6 fattyacids involves elongation of the carbon chain through the addition ofcarbon atoms and desaturation of the molecule through the addition ofdouble bonds. This requires a series of special desaturation andelongation enzymes present in the endoplasmic reticulum membrane.However, as seen in FIG. 1 and as described below, there are oftenmultiple alternate pathways for production of a specific ω-3/ω-6 fattyacid.

Specifically, all pathways require the initial conversion of oleic acidto LA, the first of the ω-6 fatty acids, by a Δ12 desaturase. Then,using the “Δ9 elongase/Δ8 desaturase pathway” and LA as substrate, longchain ω-6 fatty acids are formed as follows: (1) LA is converted to EDAby a Δ9 elongase; (2) EDA is converted to DGLA by a Δ8 desaturase; (3)DGLA is converted to ARA by a Δ5 desaturase; (4) ARA is converted to DTAby a C_(20/22) elongase; and, (5) DTA is converted to DPAn-6 by a Δ4desaturase. Alternatively, the “Δ9 elongase/Δ8 desaturase pathway” canuse ALA as substrate to produce long chain ω-3 fatty acids as follows:(1) LA is converted to ALA, the first of the ω-3 fatty acids, by a Δ15desaturase; (2) ALA is converted to ETrA by a Δ9 elongase; (3) ETrA isconverted to ETA by a Δ8 desaturase; (4) ETA is converted to EPA by a Δ5desaturase; (5) EPA is converted to DPA by a C_(20/22) elongase; and,(6) DPA is converted to DHA by a Δ4 desaturase. Optionally, ω-6 fattyacids may be converted to ω-3 fatty acids; for example, ALA is producedfrom LA by Δ15 desaturase activity; ETA and EPA are produced from DGLAand ARA, respectively, by Δ17 desaturase activity.

Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize aΔ6 desaturase and C_(18/20) elongase (i.e., the “Δ6 desaturase/Δ6elongase pathway”). More specifically, LA and ALA may be converted toGLA and STA, respectively, by a Δ6 desaturase; then, a C_(18/20)elongase converts GLA to DGLA and/or STA to ETA. Downstream PUFAs aresubsequently formed as described above.

It is contemplated that the particular functionalities required to beintroduced into a specific host organism for production of ω-3/ω-6 fattyacids will depend on the host cell (and its native PUFA profile and/ordesaturase/elongase profile), the availability of substrate, and thedesired end product(s). For example, expression of the Δ9 elongase/Δ8desaturase pathway may be preferred in some embodiments, as opposed toexpression of the Δ6 desaturase/Δ6 elongase pathway, since PUFAsproduced via the former pathway are devoid of GLA and/or STA.

One skilled in the art will be able to identify various candidate genesencoding each of the enzymes desired for ω-3/ω-6 fatty acidbiosynthesis. Useful desaturase and elongase sequences may be derivedfrom any source, e.g., isolated from a natural source (from bacteria,algae, fungi, plants, animals, etc.), produced via a semi-syntheticroute or synthesized de novo. Although the particular source of thedesaturase and elongase genes introduced into the host is not critical,considerations for choosing a specific polypeptide having desaturase orelongase activity include: (1) the substrate specificity of thepolypeptide; (2) whether the polypeptide or a component thereof is arate-limiting enzyme; (3) whether the desaturase or elongase isessential for synthesis of a desired PUFA; (4) co-factors required bythe polypeptide; and/or, (5) whether the polypeptide was modified afterits production (e.g., by a kinase or a prenyltransferase). The expressedpolypeptide preferably has parameters compatible with the biochemicalenvironment of its location in the host cell (see U.S. Pat. No.7,238,482).

It will also be useful to consider the conversion efficiency of eachparticular desaturase and/or elongase. Since each enzyme rarelyfunctions with 100% efficiency to convert substrate to product, thefinal lipid profile of unpurified oils produced in a host cell willtypically be a mixture of various PUFAs consisting of the desiredω-3/ω-6 fatty acid, as well as various upstream intermediary PUFAs.Thus, each enzyme's conversion efficiency must be considered whenoptimizing biosynthesis of a desired fatty acid.

With each of the considerations above in mind, candidate genes havingthe appropriate desaturase and elongase activities (e.g., Δ6desaturases, C_(18/20) elongases, Δ5 desaturases, Δ17 desaturases, Δ15desaturases, Δ9 desaturases, Δ12 desaturases, C_(14/16) elongases,C_(16/18) elongases, Δ9 elongases, Δ8 desaturases, Δ4 desaturases andC_(20/22) elongases) can be identified according to publicly availableliterature (e.g., GenBank), the patent literature, and experimentalanalysis of organisms having the ability to produce PUFAs. These geneswill be suitable for introduction into a specific host organism, toenable or enhance the organism's synthesis of PUFAs.

Sequence Identification of Novel Δ8 Desaturases

In the present invention, nucleotide sequences encoding Δ8 desaturaseshave been isolated from Euglena anabaena, as summarized below in Table3.

TABLE 3 Summary Of Euglena anabaena Δ8 Desaturases Nucleotide Amino AcidAbbreviation SEQ ID NO SEQ ID NO EaD8Des1 17 21 EaD8Des2 18 22 EaD8Des319 23 EaD8Des4 20 24 EaD8S 39 40 *Note: SEQ ID NO: 40 is identical insequence to SEQ ID NO: 23.

Thus, the present invention concerns an isolated polynucleotidecomprising:

-   -   (a) a nucleotide sequence encoding a polypeptide having Δ8        desaturase activity, wherein the polypeptide has at least 80%        amino acid identity, based on the Clustal V method of alignment,        when compared to an amino acid sequence as set forth in SEQ ID        NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24;    -   (b) a nucleotide sequence encoding a polypeptide having A8        desaturase activity, wherein the nucleotide sequence has at        least 80% sequence identity, based on the BLASTN method of        alignment, when compared to a nucleotide sequence as set forth        in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ        ID NO:39; or,    -   (c) a complement of the nucleotide sequence of (a) or (b),        wherein the complement and the nucleotide sequence consist of        the same number of nucleotides and are 100% complementary, and        host cells comprising the same.

In still another aspect, the invention concerns an isolatedpolynucleotide comprising a nucleotide sequence encoding a polypeptidehaving Δ8 desaturase activity, wherein the nucleotide sequence has atleast 90% sequence identity, based on the BLASTN method of alignment,when compared to a nucleotide sequence as set forth in SEQ ID NO:17, SEQID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:39.

More preferred amino acid fragments that are at least about 80%-90%identical are particularly suitable and those sequences that are atleast about 90%-95% identical are most preferred. Similarly, preferredΔ8 desaturase encoding nucleic acid sequences corresponding to theinstant ORFs are those encoding active proteins and which are at leastabout 80%-90% identical; those sequences that are at least about 90%-95%identical are most preferred.

In alternate embodiments, the instant EaD8Des1, EaD8Des2, EaD8Des3and/or EaD8Des4 desaturase sequences can be codon-optimized forexpression in a particular host organism. In general, host-preferredcodons can be determined within a particular host species of interest byexamining codon usage in proteins (preferably those expressed in thelargest amount) and determining which codons are used with highestfrequency. Then, the coding sequence for a polypeptide of interesthaving e.g., desaturase activity can be synthesized in whole or in partusing the codons preferred in the host species. All (or portions) of theDNA also can be synthesized to remove any destabilizing sequences orregions of secondary structure that would be present in the transcribedmRNA. All (or portions) of the DNA also can be synthesized to alter thebase composition to one more preferable in the desired host cell.

In one embodiment of the invention, EaD8Des3 (SEQ ID NO:19) wascodon-optimized for expression in Yarrowia lipolytica. This was possiblebased on previous determination of the Y. lipolytica codon usageprofile, identification of those codons that were preferred, anddetermination of the consensus sequence around the ‘ATG’ initiationcodon (see U.S. Pat. No. 7,238,482 and U.S. Pat. No. 7,125,672,incorporated herein by reference). The resultant synthetic gene isreferred to as EaD8S (SEQ ID NO:39). The protein sequence encoded by thecodon-optimized Δ8 desaturase gene (i.e., SEQ ID NO:40) is identical tothat of the wildtype protein sequence (i.e., SEQ ID NO:23). Similartechniques could be utilized to produce a synthetic Δ8 desaturasederived from EaD8Des1, EaD8Des2 and/or EaD8Des4 for expression in Y.lipolytica.

One skilled in the art would be able to use the teachings herein tocreate various other codon-optimized Δ8 desaturase proteins suitable foroptimal expression in alternate hosts, based on the wildtype EaD8Des1,EaD8Des2, EaD8Des3 and/or EaD8Des4 sequences. Accordingly, the instantinvention relates to any codon-optimized Δ8 desaturase protein that isderived from the wildtype nucleotide sequences of EaD8Des1 (SEQ IDNO:17), EaD8Des2 (SEQ ID NO:18), EaD8Des3 (SEQ ID NO:19) or EaD8Des4(SEQ ID NO:20). This includes, but is not limited to, the nucleotidesequence set forth in SEQ ID NO:39, which encodes a synthetic Δ8desaturase protein (i.e., EaD8S) that was codon-optimized for expressionin Yarrowia lipolytica. In alternate embodiments, it may be desirable tomodify a portion of the codons encoding EaD8Des1, EaD8Des2, EaD8Des3and/or EaD8Des4 to enhance expression of the gene in a host organismincluding, but not limited to, a plant or plant part.

Identification and Isolation of Homologs

Any of the instant desaturase sequences (i.e., EaD8Des1, EaD8Des2,EaD8Des3, EaD8Des4, EaD8S) or portions thereof may be used to search forΔ8 desaturase homologs in the same or other bacterial, algal, fungal,euglenoid or plant species using sequence analysis software. In general,such computer software matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.

Alternatively, any of the instant desaturase sequences or portionsthereof may also be employed as hybridization reagents for theidentification of Δ8 desaturase homologs. The basic components of anucleic acid hybridization test include a probe, a sample suspected ofcontaining the gene or gene fragment of interest and a specifichybridization method. Probes of the present invention are typicallysingle-stranded nucleic acid sequences that are complementary to thenucleic acid sequences to be detected. Probes are “hybridizable” to thenucleic acid sequence to be detected. Although the probe length can varyfrom 5 bases to tens of thousands of bases, typically a probe length ofabout 15 bases to about 30 bases is suitable. Only part of the probemolecule need be complementary to the nucleic acid sequence to bedetected. In addition, the complementarity between the probe and thetarget sequence need not be perfect. Hybridization does occur betweenimperfectly complementary molecules with the result that a certainfraction of the bases in the hybridized region are not paired with theproper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added (e.g., guanidinium chloride, guanidiniumthiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodiumperchlorate, rubidium tetrachloroacetate, potassium iodide, cesiumtrifluoroacetate). If desired, one can add formamide to thehybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

In additional embodiments, any of the Δ8 desaturase nucleic acidfragments described herein (or any homologs identified thereof) may beused to isolate genes encoding homologous proteins from the same orother bacterial, algal, fungal, euglenoid or plant species. Isolation ofhomologous genes using sequence-dependent protocols is well known in theart. Examples of sequence-dependent protocols include, but are notlimited to: (1) methods of nucleic acid hybridization; (2) methods ofDNA and RNA amplification, as exemplified by various uses of nucleicacid amplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor et al., Proc. Acad. Sci. U.S.A., 82:1074 (1985); or stranddisplacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and, (3) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to the Δ8desaturases described herein could be isolated directly by using all ora portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from e.g., any desired yeast or fungus usingmethodology well known to those skilled in the art (wherein thoseorganisms producing DGLA and/or ETA would be preferred). Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis,supra). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, V A; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used in PCRprotocols to amplify longer nucleic acid fragments encoding homologousgenes from DNA or RNA. PCR may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding eukaryotic genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., Proc. Natl Acad. Sci. U.S.A.,85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (Gibco/BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragmentscan be isolated (Ohara et al., Proc. Natl Acad. Sci. U.S.A., 86:5673(1989); Loh et al., Science, 243:217 (1989)).

In other embodiments, any of the Δ8 desaturase nucleic acid fragmentsdescribed herein (or any homologs identified thereof) may be used forcreation of new and/or improved fatty acid desaturases. As is well knownin the art, in vitro mutagenesis and selection, chemical mutagenesis,“gene shuffling” methods or other means can be employed to obtainmutations of naturally occurring desaturase genes (wherein suchmutations may include deletions, insertions and point mutations, orcombinations thereof). This would permit production of a polypeptidehaving desaturase activity, respectively, in vivo with more desirablephysical and kinetic parameters for function in the host cell such as alonger half-life or a higher rate of production of a desired PUFA. Ifdesired, the regions of a polypeptide of interest (i.e., a Δ8desaturase) important for enzymatic activity can be determined throughroutine mutagenesis, expression of the resulting mutant polypeptides anddetermination of their activities. An overview of these techniques isdescribed in U.S. Pat. No. 7,238,482. All such mutant proteins andnucleotide sequences encoding them that are derived from EaD8Des1,EaD8Des2, EaD8Des3, EaD8Des4 and EaD8S are within the scope of thepresent invention.

Alternatively, improved fatty acids may be synthesized by domainswapping, wherein a functional domain from any of the Δ8 desaturasenucleic acid fragments described herein are exchanged with a functionaldomain in an alternate desaturase gene to thereby result in a novelprotein. As used herein, “domain” or “functional domain” refer tonucleic acid sequence(s) that are capable of eliciting a biologicalresponse in plants or yeast.

Methods for Production of Various Omega-3 and/or Omega-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ8desaturases described herein (i.e., EaD8Des1, EaD8Des2, EaD8Des3,EaD8Des4, EaD8S or other mutant enzymes, codon-optimized enzymes orhomologs thereof), under the control of the appropriate promoters willresult in increased production of DGLA and/or ETA in the transformedhost organism. As such, the present invention encompasses a method forthe direct production of PUFAs comprising exposing a fatty acidsubstrate (i.e., EDA and/or ETrA) to the desaturase enzymes describedherein (e.g., EaD8Des1, EaD8Des2, EaD8Des3, EaD8Des4 or EaD8S), suchthat the substrate is converted to the desired fatty acid product (i.e.,DGLA and/or ETA, respectively).

More specifically, it is an object of the present invention to provide amethod for the production of DGLA in a microbial host cell (e.g., yeast,algae, bacteria, euglenoids, stramenopiles and fungi), wherein themicrobial host cell comprises:

-   -   a) a recombinant nucleotide molecule encoding a Δ8 desaturase        polypeptide having at least 80% amino acid identity, based on        the Clustal V method of alignment, when compared to a        polypeptide having an amino acid sequence selected from the        group consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23        and SEQ ID NO:24; and,    -   b) a source of EDA;        wherein the microbial host cell is grown under conditions such        that the nucleic acid fragment encoding the Δ8 desaturase is        expressed and the EDA is converted to DGLA, and wherein the DGLA        is optionally recovered.

In alternate embodiments of the present invention, the Δ8 desaturase maybe used for the conversion of ETrA to ETA. Accordingly the inventionprovides a method for the production of ETA, wherein the microbial hostcell comprises:

-   -   a) a recombinant nucleotide molecule encoding a Δ8 desaturase        polypeptide having at least 80% amino acid identity, based on        the Clustal V method of alignment, when compared to a        polypeptide having an amino acid sequence selected from the        group consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23        and SEQ ID NO:24; and,    -   b) a source of ETrA;        wherein the microbial host cell is grown under conditions such        that the nucleic acid fragment encoding the Δ8 desaturase is        expressed and the ETrA is converted to ETA, and wherein the ETA        is optionally recovered.

Alternatively, each Δ8 desaturase gene and its corresponding enzymeproduct described herein can be used indirectly for the production ofvarious ω-6 and ω-3 PUFAs (see FIG. 1 and U.S. Pat. No. 7,238,482).Indirect production of ω-3/ω-6 PUFAs occurs wherein the fatty acidsubstrate is converted indirectly into the desired fatty acid product,via means of an intermediate step(s) or pathway intermediate(s). Thus,it is contemplated that the Δ8 desaturases described herein (i.e.,EaD8Des1, EaD8Des2, EaD8Des3, EaD8Des4, EaD8S or other mutant enzymes,codon-optimized enzymes or homologs thereof) may be expressed inconjunction with additional genes encoding enzymes of the PUFAbiosynthetic pathway (e.g., Δ6 desaturases, C_(18/20) elongases, Δ17desaturases, Δ8 desaturases, Δ15 desaturases, Δ9 desaturases, Δ12desaturases, C_(14/16) elongases, C_(16/18) elongases, Δ9 elongases, Δ5desaturases, Δ4 desaturases, C_(20/22) elongases) to result in higherlevels of production of longer-chain ω-3/ω-6 fatty acids (e.g., ARA,EPA, DTA, DPAn-6, DPA and/or DHA).

In preferred embodiments, the Δ8 desaturases of the present inventionwill minimally be expressed in conjunction with a Δ9 elongase (e.g.,from Isochrysis galbana [PCT Publication No. WO 2002/077213]; fromEuglena gracilis [PCT Publication No. WO 2007/061845]; and fromEutreptiella sp. CCMP389 [PCT Publication No. WO 2007/061742]. However,the particular genes included within a particular expression cassettewill depend on the host cell (and its PUFA profile and/ordesaturase/elongase profile), the availability of substrate and thedesired end product(s).

In some embodiments, it may be useful to express more than one Δ8desaturase (i.e., the same or different Δ8 desaturase), to minimizeby-product fatty acids. The relative abundance of by-product fatty acidscould be decreased by increasing total Δ8 desaturase activity. Oneapproach to minimize by-product fatty acids would be to express morethan one Δ8 desaturase. For instance, the presence of sciadonic acid(SCI) and/or juniperonic acid (JUP) [commonly found in the seed lipidsof gymnosperms (Wolff et al., Lipids, 35(1):1-22 (2000)), such as thosein the Pinaceae family (pine)] might be considered by-product fattyacids of a Δ6 desaturase/Δ6 elongase pathway or Δ9 elongase/Δ8desaturase pathway. Although these fatty acids are considered to havevarious health-enhancing properties themselves (Nakane et al., Biol.Pharm. Bull., 23: 758-761 (2000)), their presence as by-product fattyacids in an engineered PUFA pathway, such as in an oilseed crop, may notbe desirable depending on the application.

Occasionally, a Δ6 elongase may elongate fatty acids other than theintended fatty acid. For instance, Δ6 elongases generally convert GLA toDGLA but some Δ6 elongases may also convert unintended substrates suchas LA or ALA to EDA or ETrA, respectively. In a Δ6 desaturase/Δ6elongase pathway, EDA and ETrA would be considered “by-product fattyacids”. Addition of a Δ8 desaturase to a Δ6 desaturase/Δ6 elongasepathway would provide a means to convert the “by-product fatty acids”EDA and ETrA back into the “intermediate fatty acids” DGLA and ETA,respectively.

Microbial Expression Systems, Cassettes and Vectors

The Δ8 desaturase genes and gene products described herein (i.e.,EaD8Des1, EaD8Des2, EaD8Des3, EaD8Des4, EaD8S or other mutant enzymes,codon-optimized enzymes or homologs thereof) may be expressed inheterologous microbial host cells, particularly in the cells ofoleaginous yeasts (e.g., Yarrowia lipolytica).

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors (e.g., constructs, plasmids) and DNA expression cassettes usefulfor the transformation of suitable microbial host cells are well knownin the art. The specific choice of sequences present in the construct isdependent upon the desired expression products (supra), the nature ofthe host cell and the proposed means of separating transformed cellsversus non-transformed cells. Typically, however, the vector contains atleast one expression cassette, a selectable marker and sequencesallowing autonomous replication or chromosomal integration. Suitableexpression cassettes comprise a region 5′ of the gene that controlstranscription (e.g., a promoter), the gene coding sequence, and a region3′ of the DNA fragment that controls transcriptional termination (i.e.,a terminator). It is most preferred when both control regions arederived from genes from the transformed microbial host cell, although itis to be understood that such control regions need not be derived fromthe genes native to the specific species chosen as a production host.

Transcriptional control regions (also initiation control regions orpromoters) which are useful to drive expression of the instant Δ8desaturase ORFs in the desired microbial host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter (i.e.,native, synthetic, or chimeric) capable of directing expression of thesegenes in the selected host cell is suitable for the present invention,although transcriptional and translational regions from the host speciesare particularly useful. Expression in a microbial host cell can beaccomplished in an induced or constitutive fashion. Induced expressioncan be accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, while constitutive expressioncan be achieved by the use of a constitutive promoter operably linked tothe gene of interest. As an example, when the host cell is yeast,transcriptional and translational regions functional in yeast cells areprovided, particularly from the host species (e.g., see PatentPublication No. US-2006-0115881-A1, for preferred transcriptionalinitiation regulatory regions for use in Yarrowia lipolytica). Any oneof a number of regulatory sequences can be used, depending upon whetherconstitutive or induced transcription is desired, the efficiency of thepromoter in expressing the ORF of interest, the ease of construction andthe like.

Nucleotide sequences surrounding the translational initiation codon‘ATG’ have been found to affect expression in yeast cells. If thedesired polypeptide is poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Termination controlregions may also be derived from various genes native to the preferredhosts. In alternate embodiments, the 3′-region can also be synthetic, asone of skill in the art can utilize available information to design andsynthesize a 3′-region sequence that functions as a transcriptionterminator. Optionally, a termination site may be unnecessary; however,it is most preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation andsecretion from the microbial host cell. More specifically, some of themolecular features that have been manipulated to control gene expressioninclude: the nature of the relevant transcriptional promoter andterminator sequences; the number of copies of the cloned gene (whereinadditional copies may be cloned within a single expression constructand/or additional copies may be introduced into the host cell byincreasing the plasmid copy number or by multiple integration of thecloned gene into the genome); whether the gene is plasmid-borne orintegrated into the genome of the host cell; the final cellular locationof the synthesized foreign protein; the efficiency of translation andcorrect folding of the protein in the host organism; the intrinsicstability of the mRNA and protein of the cloned gene within the hostcell; and, the codon usage within the cloned gene, such that itsfrequency approaches the frequency of preferred codon usage of the hostcell. Each of these types of modifications are encompassed in thepresent invention, as means to further optimize expression of the Δ8desaturases described herein.

Transformation of Microbial Host Cells

Once a DNA cassette that is suitable for expression in an appropriatemicrobial host cell has been obtained (e.g., a chimeric gene comprisinga promoter, ORF and terminator), it is placed in a plasmid vectorcapable of autonomous replication in a host cell, or it is directlyintegrated into the genome of the host cell. Integration of expressioncassettes can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination within the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene(s) of interest may be introduced into amicrobial host cell by any standard technique. These techniques includetransformation (e.g., lithium acetate transformation [Methods inEnzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact,electroporation, microinjection, or any other method that introduces thegene(s) of interest into the host cell.

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed”, “transformant” or “recombinant” herein. Thetransformed host will have at least one copy of the expression constructand may have two or more, depending upon whether the expression cassetteis integrated into the genome or is present on an extrachromosomalelement having multiple copy numbers.

The transformed host cell can be identified by various selectiontechniques, as described in U.S. Pat. Nos. 7,238,482 and 7,259,255 andPCT Publication No. WO 2006/052870.

Following transformation, substrates suitable for the instant Δ8desaturases (and, optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or they may be provided exogenously.

Preferred Microbial Host Cells for Recombinant Expression

Microbial host cells for expression of the instant genes and nucleicacid fragments may include hosts that grow on a variety of feedstocks,including simple or complex carbohydrates, fatty acids, organic acids,oils and alcohols, and/or hydrocarbons over a wide range of temperatureand pH values. The genes described in the instant invention have beenexpressed in an oleaginous yeast (an oleaginous stain of Yarrowialipolytica); however, it is contemplated that because transcription,translation and the protein biosynthetic apparatus are highly conserved,any bacteria, yeast, algae, euglenoid, stramenopiles and/or fungus willbe a suitable microbial host for expression of the present nucleic acidfragments.

Preferred microbial hosts are oleaginous organisms, such as oleaginousyeasts. These organisms are naturally capable of oil synthesis andaccumulation, wherein the oil can comprise greater than about 25% of thecellular dry weight, more preferably greater than about 30% of thecellular dry weight, and most preferably greater than about 40% of thecellular dry weight. Genera typically identified as comprising strainsthat may be oleaginous include, but are not limited to: Yarrowia,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon andLipomyces. More specifically, illustrative oil-synthesizing yeastsinclude: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus,Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporonpullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowialipolytica (formerly classified as Candida lipolytica). In alternateembodiments, oil biosynthesis may be genetically engineered such thatthe microbial host cell (e.g., a yeast) can produce more than 25% oil ofthe cellular dry weight, and thereby be considered oleaginous.

Preferred oleaginous yeasts are oleaginous strains of Yarrowialipolytica where particularly preferred are the Y. lipolytica strainsdesignated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.,82(1):43-9 (2002)).

Specific teachings applicable for transformation of oleaginous yeasts(i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741, U.S. Pat.No. 5,071,764 and Chen, D. C. et al. (Appl. Microbiol. Biotechnol.,48(2):232-235 (1997)). Specific teachings applicable for engineeringARA, EPA and DHA production in Y. lipolytica are provided in U.S. patentapplication Ser. No. 11/264784, U.S. patent application Ser. No.11/265761, and U.S. patent application Ser. No. 11/264737 respectively.

The preferred method of expressing genes in this yeast is by integrationof linear DNA into the genome of the host; and, integration intomultiple locations within the genome can be particularly useful whenhigh level expression of genes are desired [e.g., in the Ura3 locus(GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank AccessionNo. AF260230), the Lys5 gene locus (GenBank Accession No. M34929), theAco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus(Pox3: GenBank Accession No. XP_(—)503244; or, Aco3: GenBank AccessionNo. AJ001301), the Δ12 desaturase gene locus (U.S. Pat. No. 7,214,491),the Lip1 gene locus (GenBank Accession No. Z50020), the Lip2 gene locus(GenBank Accession No. AJ012632), and/or the Pex10 gene locus (GenBankAccession No. CAG81606.

Preferred selection methods for use in Yarrowia lipolytica areresistance to kanamycin, hygromycin and the amino glycoside G418, aswell as ability to grow on media lacking uracil, leucine, lysine,tryptophan or histidine. Additionally 5-fluoroorotic acid(5-fluorouracil-6-carboxylic acid monohydrate; “5-FOA”) may be used forselection of yeast Ura⁻ mutants. The compound is toxic to yeast cellsthat possess a functioning URA3 gene encoding orotidine 5′-monophosphatedecarboxylase (OMP decarboxylase) and, because of this toxicity, 5-FOAis especially useful for the selection and identification of Ura⁻ mutantyeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System,Oxford University: New York, v. 7, pp 109-147, 1997; see also PCTPublication No. WO 2006/052870 for 5-FOA use in Yarrowia).

An alternate preferred selection method for use in Yarrowia relies on adominant, non-antibiotic marker for Yarrowia lipolytica based onsulfonylurea (chlorimuron ethyl; E. I. duPont de Nemours & Co., Inc.,Wilmington, Del.) resistance. More specifically, the marker gene is anative acetohydroxyacid synthase (AHAS or acetolactate synthase; E.C.4.1.3.18) that has a single amino acid change (W497L) that conferssulfonyl urea herbicide resistance (PCT Publication No. WO 2006/052870).AHAS is the first common enzyme in the pathway for the biosynthesis ofbranched-chain amino acids (i.e., valine, leucine, isoleucine) and it isthe target of the sulfonylurea and imidazolinone herbicides.

Other preferred microbial hosts include oleaginous bacteria, algae,euglenoids, stramenopiles and other fungi, may of which may begenetically engineered for the production of omega-3 fatty acids. Thus,for example, transformation of Mortierella alpina (which is commerciallyused for production of ARA) with any of the present Δ8 desaturase genesunder the control of inducible or regulated promoters could yield atransformant capable of synthesizing increased quantities of DGLA. Themethod of transformation of M. alpina is described by Mackenzie et al.(Appl. Environ. Microbiol., 66:4655 (2000)). Similarly, methods fortransformation of Thraustochytriales microorganisms (e.g.,Thraustochytrium, Schizochytrium) are disclosed in U.S. Pat. No.7,001,772.

Irrespective of the host selected for expression of the Δ8 desaturasesdescribed herein, it may be necessary to screen multiple transformantsto obtain a strain displaying the desired expression level and pattern.Such screening may be accomplished by Southern analysis of DNA blots(Southern, J. Mol. Biol., 98:503 (1975)), Northern analysis of mRNAexpression (Kroczek, J. Chromatogr. Biomed. Appl., 618(1-2):133-145(1993)), Western and/or Elisa analyses of protein expression, phenotypicanalysis or GC analysis of the PUFA products.

Accordingly the scope of the present invention includes a method ofproducing either DGLA or ETA, respectively, comprising:

-   -   (a) providing an oleaginous yeast (e.g., Yarrowia lipolytica)        comprising:        -   (i) a first recombinant nucleotide molecule encoding a Δ8            desaturase polypeptide, operably linked to at least one            regulatory sequence; and,        -   (ii) a source of desaturase substrate consisting of EDA            and/or ETrA, respectively; and,    -   (b) growing the yeast of step (a) in the presence of a suitable        fermentable carbon source wherein the gene encoding the Δ8        desaturase polypeptide is expressed and EDA is converted to DGLA        and/or ETrA is converted to ETA, respectively; and,    -   (c) optionally recovering the DGLA and/or ETA, respectively, of        step (b). Substrate feeding may be required.

The nucleotide sequence of the gene encoding a Δ8 desurase may beselected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19 and SEQ ID NO:20. In alternate embodiments, the nucleotidesequence of the gene encoding a Δ8 desaturase polypeptide is set forthin SEQ ID NO:39 (wherein at least 208 codons have been optimized forexpression in Yarrowia relative to SEQ ID NO:19).

Since naturally produced PUFAs in oleaginous yeast are limited to 18:2fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA),the oleaginous yeast will be genetically engineered to express multipleenzymes necessary for long-chain PUFA biosynthesis (thereby enablingproduction of e.g., ARA, EPA, DPA and DHA), in addition to the Δ8desaturases described herein. Specifically, in one embodiment thisinvention concerns an oleaginous yeast comprising:

(a) a first recombinant DNA construct comprising an isolatedpolynucleotide encoding a Δ8 desaturase polypeptide, operably linked toat least one regulatory sequence; and,

(b) at least one additional recombinant DNA construct comprising anisolated polynucleotide, operably linked to at least one regulatorysequence, encoding a polypeptide selected from the group consisting of:a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ9 desaturase, aΔ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 elongase, aC_(14/16) elongase, a C_(16/18) elongase, a C_(18/20) elongase and aC_(20/22) elongase.

In particularly preferred embodiments, the at least one additionalrecombinant DNA construct encodes a polypeptide having delta-9 elongaseactivity.

Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesisin Microbes

Knowledge of the sequences of the present Δ8 desaturases will be usefulfor manipulating ω-3 and/or ω-6 fatty acid biosynthesis in various hostcells. Methods for manipulating biochemical pathways are well known tothose skilled in the art; and, it is expected that numerousmanipulations will be possible to maximize ω-3 and/or ω-6 fatty acidbiosynthesis in oleaginous yeasts, and particularly, in Yarrowialipolytica. This manipulation may require metabolic engineering directlywithin the PUFA biosynthetic pathway or additional manipulation ofpathways that contribute carbon to the PUFA biosynthetic pathway.Methods useful for up-regulating desirable biochemical pathways anddown-regulating undesirable biochemical pathways are well known to thoseskilled in the art. For example, biochemical pathways competing with theω-3 and/or ω-6 fatty acid biosynthetic pathways for energy or carbon, ornative PUFA biosynthetic pathway enzymes that interfere with productionof a particular PUFA end-product, may be eliminated by gene disruptionor down-regulated by other means (e.g., antisense mRNA).

Detailed discussion of manipulations within the PUFA biosyntheticpathway as a means to increase ARA, EPA or DHA (and associatedtechniques thereof) are presented in U.S. Patent Publication No.2006-0094092-A1, U.S. Patent Publication No. 2006-0115881-A1, and U.S.Patent Publication No. 2006-0110806-A1, respectively, as are desirablemanipulations in the TAG biosynthetic pathway and the TAG degradationpathway (and associated techniques thereof).

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the strategies described above. For example, the presentinvention provides methods whereby genes encoding key enzymes in the Δ9elongase/Δ8 desaturase biosynthetic pathway are introduced intooleaginous yeasts for the production of ω-3 and/or ω-6 fatty acids. Itwill be particularly useful to express the present the Δ8 desaturasegenes in oleaginous yeasts that do not naturally possess ω-3 and/or ω-6fatty acid biosynthetic pathways and coordinate the expression of thesegenes, to maximize production of preferred PUFA products using variousmeans for metabolic engineering of the host organism.

Microbial Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions thatoptimize expression of chimeric desaturase and elongase genes andproduce the greatest and most economical yield of desired PUFAs. Ingeneral, media conditions that may be optimized include the type andamount of carbon source, the type and amount of nitrogen source, thecarbon-to-nitrogen ratio, the amount of different mineral ions, theoxygen level, growth temperature, pH, length of the biomass productionphase, length of the oil accumulation phase and the time and method ofcell harvest. Microorganisms of interest, such as oleaginous yeast(e.g., Yarrowia lipolytica) are generally grown in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)) or a defined minimal mediathat lacks a component necessary for growth and thereby forces selectionof the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources are taught in U.S. Pat. No.7,238,482. Although it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing sources, preferred carbon sources are sugars,glycerol, and/or fatty acids. Most preferred is glucose and/or fattyacids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the oleaginoushost and promotion of the enzymatic pathways necessary for PUFAproduction. Particular attention is given to several metal ions (e.g.,Fe⁺², Cu⁺², Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids andPUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle andR. Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the transformant hostcells will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica).This approach is described in U.S. Pat. No. 7,238,482, as are varioussuitable fermentation process designs (i.e., batch, fed-batch andcontinuous) and considerations during growth.

Purification and Processing of PUFA Oils

PUFAs may be found in the host microorganisms as free fatty acids or inesterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids, and may be extracted from the host cells through a varietyof means well-known in the art. One review of extraction techniques,quality analysis and acceptability standards for yeast lipids is that ofZ. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). Abrief review of downstream processing is also available by A. Singh andO. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).

In general, means for the purification of PUFAs may include extraction(e.g., U.S. Pat. No. 6,797,303 and U.S. Pat. No. 5,648,564) with organicsolvents, sonication, supercritical fluid extraction (e.g., using carbondioxide), saponification and physical means such as presses, orcombinations thereof. One is referred to the teachings of U.S. Pat. No.7,238,482 for additional details.

PUFA-Containing Oils for Use in Foodstuffs, Health Food Products,Pharmaceuticals and Animal Feeds

The market place currently supports a large variety of food and feedproducts, incorporating ω-3 and/or ω-6 fatty acids (particularly e.g.,ALA, GLA, ARA, EPA, DPA and DHA). It is contemplated that the microbialbiomass comprising long-chain PUFAs, partially purified microbialbiomass comprising PUFAs, purified microbial oil comprising PUFAs,and/or purified PUFAs will function in food and feed products to impartthe health benefits of current formulations. More specifically, oils ofthe invention containing ω-3 and/or ω-6 fatty acids will be suitable foruse in a variety of food and feed products including, but not limitedto: food analogs, meat products, cereal products, baked foods, snackfoods and dairy products (see Patent Publication No. US-2006-0094092 fordetails).

Additionally, the present compositions may be used in formulations toimpart health benefit in medical foods including medical nutritionals,dietary supplements, infant formula as well as pharmaceutical products.One of skill in the art of food processing and food formulation willunderstand how the amount and composition of the present oils may beadded to the food or feed product. Such an amount will be referred toherein as an “effective” amount and will depend on the food or feedproduct, the diet that the product is intended to supplement or themedical condition that the medical food or medical nutritional isintended to correct or treat.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and, 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience, Hoboken, N.J. (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified. E. coli strains were typically grownat 37° C. on Luria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). DNA sequence was generated on an ABI Automaticsequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP272,007) using a combination of vector and insert-specific primers.Sequence editing was performed in Sequencher (Gene Codes Corporation,Ann Arbor, Mich.). All sequences represent coverage at least two timesin both directions. Comparisons of genetic sequences were accomplishedusing DNASTAR software (DNASTAR Inc., Madison, Wis.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” or “hr” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Nomenclature for Expression Cassettes:

The structure of an expression cassette will be represented by a simplenotation system of “X::Y::Z”, wherein X describes the promoter fragment,Y describes the gene fragment, and Z describes the terminator fragment,which are all operably linked to one another.

Transformation and Cultivation of Yarrowia lipolytica:

Yarrowia lipolytica strains with ATCC Accession Nos. #20362, #76982 and#90812 were purchased from the American Type Culture Collection(Rockville, Md.). Yarrowia lipolytica strains were typically grown at28-30° C. in several media, according to the recipes shown below. Agarplates were prepared as required by addition of 20 g/L agar to eachliquid media, according to standard methodology.

-   YPD agar medium (per liter): 10 g of yeast extract [Difco], 20 g of    Bacto peptone [Difco]; and 20 g of glucose.-   Basic Minimal Media (MM) (per liter): 20 g glucose; 1.7 g yeast    nitrogen base without amino acids; 1.0 g proline; and pH 6.1 (not    adjusted).-   Minimal Media+Leucine (MM+leucine or MMLeu) (per liter): Prepare MM    media as above and add 0.1 g leucine.-   Minimal Media+Leucine+Uracil (MMLeuUra) (per liter): Prepare MM    media as above and add 0.1 g leucine, 0.1 g uracil and 0.1 g    uridine.-   Minimal Media+5-Fluoroorotic Acid (MM+5-FOA) (per liter): 20 g    glucose, 6.7 g Yeast Nitrogen base, 75 mg uracil, 75 mg uridine and    appropriate amount of FOA (Zymo Research Corp., Orange, Calif.),    based on FOA activity testing against a range of concentrations from    100 mg/L to 1000 mg/L (since variation occurs within each batch    received from the supplier).-   High Glucose Media (HGM) (per liter): 80 glucose, 2.58 g KH₂PO₄ and    5.36 g K₂HPO₄, pH 7.5 (do not need to adjust).

Transformation of Yarrowia lipolytica was performed according to themethod of Chen, D. C. et al. (Appl. Microbiol. Biotechnol.,48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia wasstreaked onto a YPD plate and grown at 30° C. for approximately 18 h.Several large loopfuls of cells were scraped from the plate andresuspended in 1 mL of transformation buffer, comprising: 2.25 mL of 50%PEG, average MW 3350; 0.125 mL of 2 M lithium acetate, pH 6.0; 0.125 mLof 2 M DTT; and (optionally) 50 μg sheared salmon sperm DNA. Then,approximately 500 ng of linearized plasmid DNA (or 100 ng circularplasmid) was incubated in 100 μL of resuspended cells, and maintained at39° C. for 1 h with vortex mixing at 15 min intervals. The cells wereplated onto selection media plates and maintained at 30° C. for 2 to 3days.

Fatty Acid Analysis of Yarrowia lipolytica:

Unless otherwise stated, for fatty acid analysis cells were collected bycentrifugation and lipids were extracted as described in Bligh, E. G. &Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acidmethyl esters were prepared by transesterification of the lipid extractwith sodium methoxide (Roughan, G. and Nishida I., Arch BiochemBiophys., 276(1):38-46 (1990)) and subsequently analyzed with aHewlett-Packard 6890 GC fitted with a 30 m×0.25 mm (i.d.) HP-INNOWAX(Hewlett-Packard) column. The oven temperature was from 170° C. (25 minhold) to 185° C. at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μL of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μL hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Construction of Yarrowia lipolytica Strain Y4001U:

Y. lipolytica strain Y4001U was used as the host in Example 7, infra.The following description is a summary of the construction of strainY4001U, derived from Yarrowia lipolytica ATCC #20362, capable ofproducing about 17% EDA relative to the total lipids via expression of aΔ9 elongase/Δ8 desaturase pathway and having a Leu- and Ura-phenotype(FIG. 2A).

The development of strain Y4001U required the construction of strainY2224 (a FOA resistant mutant from an autonomous mutation of the Ura3gene of wildtype Yarrowia strain ATCC #20362) and strain Y4001(producing 17% EDA with a Leu-phenotype).

Generation Of Strain Y2224: Strain Y2224 was isolated in the followingmanner: Yarrowia lipolytica ATCC #20362 cells from a YPD agar plate werestreaked onto a MM plate (75 mg/L each of uracil and uridine, 6.7 g/LYNB with ammonia sulfate, without amino acids, and 20 g/L glucose)containing 250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28°C. and four of the resulting colonies were patched separately onto MMplates containing 200 mg/mL 5-FOA and MM plates lacking uracil anduridine to confirm uracil Ura3 auxotrophy.

Generation Of Strain Y4001 To Produce About 17% EDA Of Total Lipids:Strain Y4001 was created via integration of construct pZKLeuN-29E3 (FIG.2B). This construct, comprising four chimeric genes (i.e., a Δ12desaturase, a C_(16/18) elongase and two Δ9 elongases), was integratedinto the Leu2 loci of strain Y2224 to thereby enable production of EDA.

Construct pZKLeuN-29E3 contained the components shown below in Table 4.

TABLE 4 Description of Plasmid pZKLeuN-29E3 (SEQ ID NO: 42) RE Sites AndNucleotides Within SEQ ID Description Of NO: 42 Fragment And ChimericGene Components BsiW I/Asc I 788 bp 3′ portion of Yarrowia Leu2 gene(GenBank (7797-7002) Accession No. AF260230) Sph I/Pac I 703 bp 5′portion of Yarrowia Leu2 gene (GenBank (4302-3591) Accession No.AF260230) Swa I/BsiW I GPD::FmD12::Pex20, comprising: (10533-7797) GPD:Yarrowia lipolytica GPD promoter (U.S. Pat. No. 7,259,255); FmD12:Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO: 43) (labeled as“F.D12” in Figure; PCT Publication No. WO 2005/047485); Pex20: Pex20terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.AF054613) Bgl II/Swa I EXP1::EgD9eS::Lip1, comprising: (12559-10533)EXP1: Yarrowia lipolytica export protein (EXP1) promoter (labeled as“Exp pro” in Figure; PCT Publication No. WO 2006/052870 and U.S. PatentApplication No. 11/265761); EgD9eS: codon-optimized Δ9 elongase (SEQ IDNO: 45), derived from Euglena gracilis (labeled as “EgD9E” in Figure;PCT Publication No. WO 2007/061742); Lip1: Lip1 terminator sequence fromYarrowia Lip1 gene (GenBank Accession No. Z50020) Pme I/Cla IFBAINm::EgD9eS::Lip2, comprising: (12577-1) FBAINm: Yarrowia lipolyticaFBAINm promoter (U.S. Pat. No. 7,202,356); EgD9eS: codon-optimized Δ9elongase gene (SEQ ID NO: 45), derived from Euglena gracilis (labeled as“EgD9ES” in Figure; PCT Publication No. WO 2007/061742); Lip2: Lip2terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) Cla I/EcoR I LoxP::Ura3::LoxP, comprising: (1-1736) LoxPsequence (SEQ ID NO: 47); Yarrowia Ura3 gene (GenBank Accession No.AJ306421); LoxP sequence (SEQ ID NO: 47) EcoR I/Pac I YAT1::ME3S::Pex16,comprising: (1736-3591) YAT1: Yarrowia lipolytica YAT1 promoter (labeledas “YAT” in Figure; Patent Publication No. U.S. 2006/0094102-A1); ME3S:codon-optimized C_(16/18) elongase gene (SEQ ID NO: 48), derived from M.alpina (PCT Publication No. WO 2007/046817); Pex16: Pex16 terminatorsequence of Yarrowia Pex 16 gene (GenBank Accession No. U75433)

Plasmid pZKLeuN-29E3 was digested with AscI/SphI, and then used fortransformation of Y. lipolytica strain Y2224 (i.e., ATCC #20362 Ura3-)according to the General Methods. The transformant cells were platedonto MMLeu media plates and maintained at 30° C. for 2 to 3 days. Thecolonies were picked and streaked onto MM and MMLeu selection plates.The colonies that could grow on MMLeu plates but not on MM plates wereselected as Leu-strains. Single colonies of Leu-strains were theninoculated into liquid MMLeu at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of EDA in the transformants containingthe 4 chimeric genes of pZKLeuN-29E3, but not in the Yarrowia Y2224control strain. Most of the selected 36 Leu-strains produced about 12 to16.9% EDA of total lipids. There were 3 strains (i.e., strains #11, #30and #34) that produced about 17.4%, 17% and 17.5% EDA of total lipids;they were designated as strains Y4001, Y4002 and Y4003, respectively.

Single colonies of Y4001, Y4002 and Y4003 strains were inoculated inliquid MMLeu at 30° C. and shaken at 250 rpm/min for 2 days. The cellswere collected by centrifugation, resuspended in High Glucose Media andthen shaken at 250 rpm/min for 5 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC. GC analyses showed that the Y4001, Y4002 andY4003 strains produced about 24% EDA of total lipids.

Generation Of Strain Y4001U (Leu-, Ura-): Strain Y4001U was created viatemporary expression of the Cre recombinase enzyme in plasmid pY116(FIG. 2C) within strain Y4001 to produce a Leu- and Ura-phenotype.Construct pY116 contained the following components:

TABLE 5 Description of Plasmid pY116 (SEQ ID NO: 50) RE Sites AndNucleotides Within SEQ ID NO: 50 Description Of Fragment And ChimericGene Components 1328-448 ColE1 plasmid origin of replication 2258-1398Ampicillin-resistance gene (Amp^(R)) for selection in E. coli 3157-4461Yarrowia autonomous replication sequence (ARS18; GenBank Accession No.A17608) SwaI/PacI Yarrowia Leu2 gene (GenBank Accession No. AF260230)6667-4504 Swa I/Pme I GPAT::Cre::XPR2, comprising: (6667-218) GPAT:Yarrowia lipolytica GPAT promoter (U.S. Pat. No. 7,264,949); Cre:Enterobacteria phage P1 Cre gene for recombinase protein (GenBankAccession No. X03453); XPR2: ~100 bp of the 3′ region of the YarrowiaXpr gene (GenBank Accession No. M17741)

Plasmid pY116 was used for transformation of freshly grown Y4001 cellsaccording to the General Methods. The transformant cells were platedonto MMLeuUra plates containing 280 μg/mL sulfonylurea (chlorimuronethyl, E. I. duPont de Nemours & Co., Inc., Wilmington, Del.) andmaintained at 30° C. for 3 to 4 days. Four colonies were picked,inoculated into 3 mL liquid YPD media at 30° C. and shaken at 250rpm/min for 1 day. The cultures were diluted to 1:50,000 with liquidMMLeuUra media, and 100 μL was plated onto new YPD plates and maintainedat 30° C. for 2 days. Colonies were picked and streaked onto MMLeu andMMLeuUra selection plates. The colonies that could grow on MMLeuUraplates but not on MMLeu plates were selected and analyzed by GC toconfirm the presence of C20:2 (EDA). Several strains, having a Leu- andUra-phenotype, produced about 17% EDA of total lipids and weredesignated collectively as Y4001U; one of these strains was designatedas Y4001U1.

Example 1 Synthesis of a cDNA Library From Euglena anabaena UTEX 373

The present Example describes the synthesis of a cDNA library fromEuglena anabaena UTEX 373. This work included preparation of RNA,synthesis of cDNA, and generation of a cDNA library.

Growth of Euglena anabaena UTEX 373 and Preparation of RNA

Euglena anabaena UTEX 373 was obtained from Dr. Richard Triemer's lab atMichigan State University (East Lansing, Mich.). Approximately 2 mL ofculture was removed for lipid analysis and centrifuged at 1,800×g for 5min. The pellet was washed once with water and re-centrifuged. Theresulting pellet was dried for 5 min under vacuum, resuspended in 100 μLof trimethylsulfonium hydroxide (TMSH) and incubated at room temperaturefor 15 min with shaking. After this step, 0.5 mL of hexane was added andthe vials were incubated for 15 min at room temperature with shaking.Fatty acid methyl esters (5 μL injected from hexane layer) wereseparated and quantified using a Hewlett-Packard 6890 Gas Chromatographfitted with an Omegawax 320 fused silica capillary column (Supelco Inc.,Catalog No. 24152). The oven temperature was programmed to hold at 170°C. for 1.0 min, increase to 240° C. at 5° C./min and then hold for anadditional 1.0 min. Carrier gas was supplied by a Whatman hydrogengenerator. Retention times were compared to those for methyl esters ofstandards commercially available (Nu-Chek Prep, Inc. Catalog No. U-99-A)and the resulting chromatogram is shown in FIG. 3. The presence of EDA,ERA, EPA and DHA in the fatty acid profile, with the absence of GLA andSTA, suggested that Euglena anabaena uses the Δ9 elongase/Δ8 desaturasepathway for long-chain (LC) PUFA biosynthesis and would be a good sourcefor LC-PUFA biosynthetic genes such as, but not limited to, Δ8desaturases.

The remaining 5 mL of an actively growing culture was transferred into25 mL of AF-6 Medium (Watanabe & Hiroki, NIES-Collection List ofStrains, 5^(th) ed., National Institute for Environmental Studies,Tsukuba, 127 pp (2004)) in a 125 mL glass flask. Euglena anabaenacultures were grown at 22° C. with a 16 h light, 8 h dark cycle for 2weeks with very gentle agitation.

After 2 weeks, the culture (25 mL) was transferred to 100 mL of AF-6medium in a 500 mL glass bottle and the culture was grown for 1 month asdescribed above. After this time, two 50 mL aliquots were transferredinto two separate 500 mL glass bottles containing 250 mL of AF-6 mediumand the cultures were grown for two months as described above (giving atotal of ˜600 mL of culture). Next, the cultures were pelleted bycentrifugation at 1,800×g for 10 min, washed once with water andre-centrifuged. Total RNA was extracted from one of the resultingpellets using the RNA STAT-60™ reagent (TEL-TEST, Inc., Friendswood,Tex.) and following the manufacturer's protocol provided (use 5 mL ofreagent, dissolved RNA in 0.5 mL of water). In this way, 340 μg of totalRNA (680 ug/mL) was obtained from the pellet. The remaining pellet wasfrozen in liquid nitrogen and stored at −80° C. The mRNA was isolatedfrom all 340 μg of total RNA using the mRNA Purification Kit (AmershamBiosciences, Piscataway, N.J.) following the manufacturer's protocolprovided. In this way, 9.0 μg of mRNA was obtained.

Preparation of Euglena anabaena cDNA and Generation of cDNA Libraryeug1c

A cDNA library was generated using the Cloneminer™ cDNA LibraryConstruction Kit (Catalog No. 18249-029, Invitrogen Corporation,Carlsbad, Calif.) and following the manufacturer's protocol provided(Version B, 25-0608). Using the non-radiolabeling method, cDNA wassynthesized from 5.12 μg of mRNA (described above) using theBiotin-attB2-Oligo(dT) primer. After synthesis of the first and secondstrand, the attB1 adapter was added, ligated and the cDNA was sizefractionated using column chromatography. DNA from fractions wereconcentrated, recombined into PDONR™222 and transformed into E. coliElectroMAX™ DH10B™ T1 Phage-Resistant cells (Invitrogen Corporation).The Euglena anabaena library was named eug1c.

The cDNA library eug1c was plated onto LB+Kanamycin plates (approx.100,000 colonies), the colonies were scraped off and DNA was isolatedusing the QIAprep® Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.)following the manufacturer's protocol. In this way, a plasmid DNAsub-library from eug1c was obtained.

Example 2 Isolation of a cDNA Fragment Encoding a Partial Δ8 DesaturaseFrom Euglena anabaena UTEX 373

The present Example describes the identification of a cDNA fragment (SEQID NO:1) encoding a partial Δ8 desaturase from Euglena anabaena UTEX373, based on PCR amplification of the cDNA library using degenerateoligonucleotides based on a Euglena gracilis Δ8 desaturase sequence (SEQID NO:2).

Identification of cDNA Fragments Encoding Partial Putative Δ8Desaturases

The plasmid DNA sub-library described in Example 1 was used as templatefor degenerate PCR using degenerate primers based on a nucleotidesequence of the Euglena gracilis Δ8 fatty acid desaturase (SEQ ID NO:2;described as Eg5 in PCT Publication No. WO 2006/012325) and thevector-specific primer pDonor222Eg5-1 (SEQ ID NO:3). The 4 degenerateprimers used are shown in Table 6.

TABLE 6 Degenerate Oligonucleotides Used To Amplify A Portion Of The A8Desaturase Genes From Euglena anabaena UTEX 373 Primer NucleotideSequence SEQ ID NO: D8DEG3-1 RTTRTGNCKATCTTTCCACCA SEQ ID NO:4 D8DEG3-2RTTRTGNCKGTCTTTCCACCA SEQ ID NO:5 D8DEG3-3 RTTRTGNCKATCCTTCCACCA SEQ IDNO:6 D8DEG3-4 RTTRTGNCKGTCCTTCCACCA SEQ ID NO:7

A total of 5 reactions were set up for the cDNA sample. The reactionmixture contained 1 μL of cDNA, 1 μL each of the vector-specific anddegenerate primers (20 μM) and Phusion™ High-Fidelity DNA Polymerase(Catalog No. F553S, Finnzymes Oy, Finland). The PCR was carried outfollowing the manufacturer's protocol. The resulting DNA fragments werecloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol.

Plasmid DNA from the resulting clones was purified using the QIAprep®Spin Miniprep Kit (Qiagen Inc.) following the manufacturer's protocoland DNA inserts were end-sequenced in 384-well plates, usingvector-primed T7 primer (SEQ ID NO:8) and M13rev-28 primer (SEQ ID NO:9)with the ABI BigDye version 3 Prism sequencing kit. For the sequencingreaction, 100-200 ng of template and 6.4 pmol of primer were used, andthe following reaction conditions were repeated 25 times: 96° C. for 10sec, 50° C. for 5 sec and 60° C. for 4 min. After ethanol-based cleanup,cycle sequencing reaction products were resolved and detected onPerkin-Elmer ABI 3700 automated sequencers.

A consensus sequence was assembled from the individual sequencesobtained and one representative clone, designated as pHD23-1 (SEQ IDNO:10) having a sequence identical to the consensus was choosen forfurther study.

Identification of the partial cDNA insert in pHD23-1 (SEQ ID NO:1) as apartial Δ8 desaturase was confirmed using BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol., 215:403-410 (1993))searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL and DDBJ databases). The partial cDNA sequence obtained(SEQ ID NO:1) was translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States, Nat. Genet.,3:266-272 (1993)) provided by the NCBI with the default parameter andthe filter turned off. For convenience, the P-value (probability) ofobserving a match of a cDNA sequence to a sequence contained in thesearched databases merely by chance as calculated by BLAST are reportedherein as a “pLog” value, which represents the negative of the logarithmof the reported P-value. Accordingly, the greater the pLog value, thegreater the likelihood that the cDNA sequence and the BLAST “hit”represent homologous proteins.

The BLASTX search using the nucleotide sequence insert from pHD23-1revealed similarity of the protein encoded by the partial cDNA (SEQ IDNO:1) to the Euglena gracilis Δ8 desaturase amino acid sequence (SEQ IDNO:11) (NCBI Accession No. AAD45877 (GI 5639724), locus AAD45877, CDSAF139720; Wallis and Browse, Arch. Biochem. Biophys., 365:307-316(1999)) and yielded a pLog value of 63.4 (E value of 4e-63).

Example 3 Isolation of Full-length Δ8 Desaturases From Euglena anabaenaUTEX 373

Approximately 17,000 clones of cDNA library eug1c were plated onto threelarge square (24 cm×24 cm) petri plates (Corning, Corning, N.Y.) eachcontaining LB+50 μg/mL kanamycin agar media. Cells were grown overnightat 37° C. and plates were then cooled to room temperature.

Colony Lifts

Biodyne B 0.45 μm membrane (Catalog No. 60207, Pall Corporation,Pensacola, Fla.) was trimmed to approximately 22 cm×22 cm and themembrane was carefully layed on top of the agar to avoid air bubbles.After incubation for 2 min at room temperature, the membrane was markedfor orientation, lifted off with tweezers and placed colony-side up onfilter paper soaked with 0.5 M sodium hydroxide and 1.5 M sodiumchloride. After denaturation for 4 min, the sodium hydroxide wasneutralized by placing the membrane on filter paper soaked with 0.5 MTris-HCL (pH 7.5) and 1.5 M sodium chloride for 4 min. This step wasrepeated and the membrane was rinsed briefly in 2×SSC buffer (20×SSC is3 M sodium chloride, 0.3 M sodium citrate; pH 7.0) and air dried onfilter paper.

Hybridization

Membranes were pre-hybridized at 65° C. in 200 mL hybridization solutionfor 2 hr. Hybridization solution contained 6×SSPE (20×SSPE is 3 M sodiumchloride, 0.2 M sodium phosphate, 20 mM EDTA; pH 7.4), 5× Denhardt'sreagent (100× Denhardt's reagent is 2% (w/v) Ficoll, 2% (w/v)polyvinylpyrrolidone, 2% (w/v) acetylated bovine serum albumin), 0.5%sodium dodecyl sulfate (SDS), 100 μg/mL sheared salmon sperm DNA and 5%dextran sulfate.

A DNA probe was made using an agarose gel purified EcoRI DNA fragment,containing the Euglena anabaena Δ8 desaturase partial DNA fragment, frompHD23-1 (Example 2) labeled with P³² dCTP using the RadPrime DNALabeling System (Catalog No. 18428-011, Invitrogen, Carlsbad, Calif.)following the manufacturer's instructions. Unincorporated P³² dCTP wasseparated using a NICK column (Catalog No. 17-0855-02, AmershamBiosciences, Piscataway, N.J.) following the manufacturer'sinstructions. The probe was denatured for 5 min at 100° C., placed onice for 3 min and half was added to the hybridization solution.

The membrane was hybridized with the probe overnight at 65° C. withgentle shaking and then washed the following day twice with 2×SSCcontaining 0.5% SDS (5 min each) and twice with 0.2×SSC containing 0.1%SDS (15 min each). After washing, hyperfilm (Catalog No. RPN30K,Amersham Biosciences, Piscataway, N.J.) was exposed to the membraneovernight at −80° C.

Based on alignment of plates with the exposed hyperfilm, positivecolonies were picked using the blunt end of a Pasteur pipette into 1 mLof water and vortexed. Several dilutions were made and plated onto smallround Petri dishes (82 mm) containing LB media plus 50 μg/mL kanamycinto obtain around 100 well isolated colonies on a single plate. Liftswere done as described above except NytranN membrane circles (CatalogNo. 10416116, Schleicher & Schuell, Keene, N.H.) were used andhybridization was carried out in 100 mL using the remaining radiolabeledprobe. In this way, positive clones were confirmed.

Individual positive clones were grown at 37° C. in LB+50 μg/mL kanamycinliquid media and plasmid was purified using the QIAprep® Spin MiniprepKit (Qiagen Inc.) following the manufacturer's protocol.

The plasmid insert was sequenced as described in Example 2 with the ABIBigDye version 3 Prism sequencing kit using vector-primed T7 primer (SEQID NO:8), vector-primed M13rev-28 primer (SEQ ID NO:9) and the poly(A)tail-primed WobbleT oligonucleotides. Briefly, the WobbleT primer is anequimolar mix of 21 mer poly(T)A, poly(T)C and poly(T)G, used tosequence the 3′ end of cDNA clones. Based on initial sequence data,additional internal fragment sequence was obtained in a similar wayusing oligonucleotide EaD8seq-1 (SEQ ID NO:12). In this way, the fullinsert sequences of the eug1c Δ8 desaturase clones were obtained.

Sequences were aligned and compared using Sequencher™ (Version 4.2, GeneCodes Corporation, Ann Arbor, Mich.) and in this way, the clones couldbe categorized into one of four distinct groups based on insert sequence(designated as EaD8Des1, EaD8Des2, EaD8Des3 or EaD8Des4). Representativeclones containing the cDNA for each class of sequence were chosen forfurther study and sequences for each representative plasmid (i.e.,pLF118-1, pLF118-2, pLF118-3 and pLF118-4) are shown in SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively. The sequenceshown by a string of NNNN's represents a region of the polyA tail whichwas not sequenced. The coding sequences for EaD8Des1, EaD8Des2, EaD8Des3and EaD8Des4 are shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 andSEQ ID NO:20, respectively. The corresponding amino acid sequences forEaD8Des1, EaD8Des2, EaD8Des3 and EaD8Des4 are shown in SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively. EaD8Des1,EaD8Des2, EaD8Des3 and EaD8Des4 are collectively referred to as EaD8Desgenes.

Example 4 Primary Sequence Analysis of the Δ8 Desaturase Sequences ofEuglena anabaena UTEX 373 and Comparison to a Δ8 Desaturase Sequence ofEuglena gracilis

The amino acid sequences for EaD8Des1 (SEQ ID NO:21), EaD8Des2 (SEQ IDNO:22), EaD8Des3 (SEQ ID NO:23) and EaD8Des4 (SEQ ID NO:24) werecompared using the Clustal W method (using the MegAlign™ v 6.1 programof the LASERGENE bioinformatics computing suite (DNASTAR Inc.) with thedefault parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB).

Compared to EaD8Des1 (SEQ ID NO:21), EaD8Des2 (SEQ ID NO:22) has 3 aminoacid substitutions (i.e., T110S, M2231 and K251T; based on numbering forEaD8Des1), EaD8Des3 (SEQ ID NO:23) has 2 amino acid substitutions (i.e.,T110S and K251T) and EaD8Des4 (SEQ ID NO:24) has 1 amino acidsubstitution (i.e., T110S).

The amino acid sequences for EaD8Des1 (SEQ ID NO:21), EaD8Des2 (SEQ IDNO:22), EaD8Des3 (SEQ ID NO:23) and EaD8Des4 (SEQ ID NO:24) wereevaluated by BLASTP (Basic Local Alignment Search Tool; Altschul et al.,J. Mol. Biol., 215:403-410 (1993)) searches for similarity to sequencescontained in the BLAST “nr” database (supra, Example 2) using defaultparameters and the filter off. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value.

All four sequences yielded a pLog value of 177 (P value of e-177) versusthe Euglena gracilis Δ8 desaturase amino acid sequence (SEQ ID NO:11)(NCBI Accession No. AAD45877 (GI 5639724), locus AAD45877, CDS AF139720;Wallis and Browse, Arch. Biochem. Biophys., 365:307-316 (1999)) whencompared to the “nr” database. BLAST scores and probabilities indicatethat the instant nucleic acid fragments encode entire Euglena anabaenaΔ8 fatty acid desaturases.

The amino acid sequences for EaD8Des1 (SEQ ID NO:21), EaD8Des2 (SEQ IDNO:22), EaD8Des3 (SEQ ID NO:23) and EaD8Des4 (SEQ ID NO:24) were thencompared to a functional variant Euglena gracilis Δ8 desaturase aminoacid sequence (identified herein as EgD8 and set forth as SEQ ID NO:25;described as Eg5 in PCT Application No. WO 2006/012325) using BlastP(default parameters, filter off), Clustal V and the Jotun Hein methodsof sequence comparison and the % identity using each method is shown inTable 7.

Sequence percent identity calculations performed by the Clustal V method(Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci., 5:151-153(1989); Higgins et al., Comput. Appl. Biosci., 8:189-191 (1992)) weredone using the MegAlign™ v 6.1 program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) with the defaultparameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5 and GAP LENGTH PENALTY=10). Sequence percent identitycalculations performed by the Jotun Hein method (Hein, J. J., Meth.Enz., 183:626-645 (1990)) were done using the MegAlign™ v 6.1 program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,Wis.) with the default parameters for pairwise alignment (KTUPLE=2).

TABLE 7 Sequence Comparison Of EaD8Des1 (SEQ ID NO: 21), EaD8Des2 (SEQID NO: 22), EaD8Des3 (SEQ ID NO: 23) And EaD8Des4 (SEQ ID NO: 24) ToEgD8 (SEQ ID NO: 25) % Identity to % Identity to EgD8 EgD8 % Identity toEgD8 (SEQ ID NO: 25) (SEQ ID NO: 25) (SEQ ID NO: 25) by the Jotun Heinby the Clustal V Desaturase by BLASTP Method Method EaD8Des1 73% 74.4%72.1% EaD8Des2 73% 74.2% 71.9% EaD8Des3 73% 74.2% 71.9% EaD8Des4 73%74.2% 71.9%

The Clustal V alignment of these five amino acid sequences can be seenin FIGS. 4A, 4B and 4C. Table 8 below sets forth a comparison of thepercent identity (shown in the upper half triangle) and percentdivergence (shown in the lower half triangle), among the five Δ8desaturase sequences aligned in FIGS. 4A, 4B and 4C.

TABLE 8 Percent Identity And Percent Divergence Among EaD8Des1 (SEQ IDNO: 21), EaD8Des2 (SEQ ID NO: 22), EaD8Des3 (SEQ ID NO: 23), EaD8Des4(SEQ ID NO: 24) And EgD8 (SEQ ID NO: 25) EaD8Des1 EaD8Des2 EaD8Des3EaD8Des4 EgD8 EaD8Des1 — 99.3 99.5 99.8 72.1 EaD8Des2 0.7 — 99.8 99.571.9 EaD8Des3 0.5 0.2 — 99.8 71.9 EaD8Des4 0.2 0.5 0.2 — 71.9 EgD8 31.431.8 31.8 31.8 —

Example 5 Functional Analysis of the Euglena anabaena UTEX 373 Δ8Desaturases in Yarrowia lipolytica

The present Example describes functional analysis of EaD8Des1 (SEQ IDNO:21), EaD8Des2 (SEQ ID NO:22), EaD8Des3 (SEQ ID NO:23) and EaD8Des4(SEQ ID NO:24) in Yarrowia lipolytica. This work included the followingsteps: (1) PCR amplification of each Δ8 desaturase with appropriaterestriction sites for cloning from the plasmids described in Example 2;(2) cloning of the EaD8Des PCR products into cloning vector pCR-Blunt®(Invitrogen Corporation) to produce pY120-1, pY120-2, pY120-3 andpY120-4; (3) cloning of the EaD8Des genes into Yarrowia expressionvector pY115 to produce pY175, pY176, pY177 and pY178; and, (4)comparison of lipid profiles within transformant organisms comprisingpY175, pY176, pY177 and pY178, after substrate feeding.

PCR Amplification of the Euglena anabaena Δ8 Desaturase Genes

In order to introduce NotI and NcoI restriction sites at the 5′ end ofthe coding sequences and a NotI site at the 3′ end of the codingsequences, each of the EaD8Des genes were PCR amplified. The codingsequences for EaD8Des1 (SEQ ID NO:17), EaD8Des2 (SEQ ID NO:18), EaD8Des3(SEQ ID NO:19) and EaD8Des4 (SEQ ID NO:20) were amplified from pLF118-1(SEQ ID NO:13), pLF118-2 (SEQ ID NO:14), pLF118-3 (SEQ ID NO:15) andpLF118-4 (SEQ ID NO:16), respectively, with oligonucleotide primersEaD8-5 (SEQ ID NO:26) and EaD8-3 (SEQ ID NO:27) using the Phusion™High-Fidelity DNA Polymerase (Catalog No. F553S, Finnzymes Oy, Finland)following the manufacturer's protocol. The resulting DNA fragments werecloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol, to produce pLF120-1 (SEQ ID NO:28), pLF120-2 (SEQ ID NO:29),pLF120-3 (SEQ ID NO:30) and pLF120-4 (SEQ ID NO:31), respectively.

Construction of Yarrowia Expression Vectors pY115, pY175, pY176, pY177and pY178

Plasmid pY5-30 (described in U.S. Pat. No. 7,259,255) is a shuttleplasmid that can replicate both in E. coli and Yarrowia lipolytica.Plasmid pY5-30 contains the following: a Yarrowia autonomous replicationsequence (ARS18); a ColE1 plasmid origin of replication; anampicillin-resistance gene (AmpR), for selection in E. coli; a YarrowiaLEU2 gene, for selection in Yarrowia; and a chimeric TEF::GUS::XPR gene.Plasmid pDMW263 (SEQ ID NO:32) was created from pY5-30, by replacing theTEF promoter with the Yarrowia lipolytica FBAINm promoter (U.S. Pat. No.7,202,356) using techniques well known to one skilled in the art.Briefly, this promoter refers to a modified promoter which is located inthe 5′ upstream untranslated region in front of the ‘ATG’ translationinitiation codon of the fructose-bisphosphate aldolase enzyme (E.C.4.1.2.13) encoded by the fbal gene and that is necessary for expression,plus a portion of 5′ coding region that has an intron, wherein FBAINmhas a 52 bp deletion between the ATG translation initiation codon andthe intron of the FBAIN promoter (thereby including only 22 amino acidsof the N-terminus) and a new translation consensus motif after theintron. Table 9 summarizes the components of pDMW263 (SEQ ID NO:32).

TABLE 9 Components Of Plasmid pDMW263 RE Sites and Nucleotides WithinDescription of Fragment and SEQ ID NO: 32 Chimeric Gene Components4992-4296 ARS18 sequence (GenBank Accession No. A17608) SaII/SacIIFBAINm::GUS::XPR, comprising: (8505-2014) FBAINm: Yarrowia lipolyticaFBAINm promoter (U.S. Pat. No. 7,202,356); GUS: E. coli gene encodingβ-glucuronidase (Jefferson, R. A. Nature, 14: 342: 837-838 (1989); XPR:~100 bp of the 3′ region of the Yarrowia Xpr gene (GenBank Accession No.M17741) 6303-8505 Yarrowia Leu2 gene (GenBank Accession No. AF260230)

The NcoI/SalI DNA fragment from pDMW263 (SEQ ID NO:32), containing theYarrowia lipolytica FBAINm promoter, was cloned into the NcoI/SalI DNAfragment of pDMW237 (SEQ ID NO:33), previously described in PCTPublication No. WO 2006/012325 (the contents of which are herebyincorporated by reference), containing a synthetic Δ9 elongase genederived from Isochrysis galbana and codon-optimized for expression inYarrowia lipolytica, to produce pY115 (SEQ ID NO:34; FIG. 5A). In FIG.5A and FIG. 5B, the modified FBAINm promoter is labeled as FBA1+Intron.

The NcoI/NotI DNA fragments from pLF120-1 (SEQ ID NO:28), pLF120-2 (SEQID NO:29), pLF120-3 (SEQ ID NO:30) and pLF120-4 (SEQ ID NO:31),containing each EaD8Des, were cloned into the NcoI/NotI DNA fragmentfrom pY115, containing the Yarrowia lipolytica FBAINm promoter, toproduce pY175 (SEQ ID NO:35; FIG. 5B), pY176 (SEQ ID NO:36), pY177 (SEQID NO:37) and pY178 (SEQ ID NO:38), respectively.

Functional Analysis of the Euglena anabaena Δ8 Desaturase Genes inYarrowia lipolytica Strain Y2224

Strain Y2224 (see General Methods) was transformed with pY175 (SEQ IDNO:35), pY176 (SEQ ID NO:36), pY177 (SEQ ID NO:37) and pY178 (SEQ IDNO:38) as described in the General Methods.

Single colonies of transformant Yarrowia lipolytica containing pY175,pY176, pY177 and pY178 were grown in 3 mL minimal media lacking uracilsupplemented with 0.2% tergitol at 30° C. for 1 day. After this, 0.1 mLwas transferred to 3 mL of the same medium supplemented with EDA(20:2(11,14)) or ETrA (20:3(11,14,17)) to 0.175 mM. These were incubatedfor 16 hr at 30° C., 250 rpm and then pellets were obtained bycentrifugation. Cells were washed once with water, pelleted bycentrifugation and air dried. Pellets were transesterified (Roughan, G.and Nishida, I., Arch. Biochem. Biophys., 276(1):38-46 (1990)) with 500μL of 1% sodium methoxide for 30 min at 50° C. after which 500 μL of 1 Msodium chloride and 100 μL of heptane were added. After thorough mixingand centrifugation, fatty acid methyl esters (FAMEs) were analyzed byGC. FAMEs (5 μL injected from hexane layer) were separated andquantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with anOmegawax 320 fused silica capillary column (Catalog No. 24152, SupelcoInc.). The oven temperature was programmed to hold at 220° C. for 2.6min, increase to 240° C. at 20° C./min and then hold for an additional2.4 min. Carrier gas was supplied by a Whatman hydrogen generator.Retention times were compared to those for methyl esters of standardscommercially available (Nu-Chek Prep, Inc.).

The fatty acid profiles for Yarrowia lipolytica expressing pY175, pY176,pY177 and pY178 are shown in FIG. 6. Percent C20 desaturation (“C20%delta-8 desat”) was calculated either by dividing the weight percent (wt%) for DGLA by the sum of the wt % for EDA and DGLA and multiplying by100 to express as a % or by dividing the wt % for ETA by the sum of thewt % for ERA and DTA and multiplying by 100 to express as a %, dependingon which substrate was fed (i.e., EDA or ERA). Averages are indicated byAve. followed by the appropriate header. The ratio of desaturation ofEDA to ERA is calculated by dividing the Ave. C20% delta-8 desat for EDAby that of ERA.

All of the Euglena anabaena Δ8 desaturases function similarly well inYarrowia and convert approximately 50% of the fed EDA to DGLA. Thereappears to be a slight preference for the EDA over ERA with a EDA/ERAratio of 1.1 to 1.2.

Example 6 Synthesis of a Codon-Optimized Δ8 Desaturase Gene for Yarrowialipolytica (EaD8S)

The codon usage of the Δ8 desaturase gene (EaD8Des3; SEQ ID NO:19) ofEuglena anabaena was optimized for expression in Yarrowia lipolytica, ina manner similar to that described in PCT Publication No. WO 2004/101753and U.S. Pat. No. 7,125,672. Specifically, a codon-optimized Δ8desaturase gene (designated “EaD8S”, SEQ ID NO:39) was designed based onthe coding sequence of EaD8Des3 (SEQ ID NOs:19 and 23), according to theYarrowia codon usage pattern (PCT Publication No. WO 2004/101753), theconsensus sequence around the ‘ATG’ translation initiation codon, andthe general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene,265(1-2):11-23 (2001)). In addition to modification of the translationinitiation site, 231 bp of the 1260 bp coding region were modified(18.3%) and 208 codons were optimized (49.5%). The GC content wasreduced from 56.8% within the wild type gene (i.e., EaD8Des3) to 54.8%within the synthetic gene (i.e., EaD8S). A NcoI site and NotI sites wereincorporated around the translation initiation codon and after the stopcodon of EaD8S (SEQ ID NO:39), respectively. FIGS. 7A and 7B show acomparison of the nucleotide sequences of EaD8Des3 (SEQ ID NO:19) andEaD8S (SEQ ID NO:39). The protein sequence encoded by thecodon-optimized gene (i.e., SEQ ID NO:40) is identical to that of thewildtype EaD8Des3 protein sequence (i.e., SEQ ID NO:23). The designedEaD8S gene was synthesized by GenScript Corporation (Piscataway, N.J.)and cloned into pUC57 (GenBank Accession No. Y14837) to generate pEaD8S(SEQ ID NO:41; FIG. 8A).

Example 7 Construction and Functional Analysis of Yarrowia lipolyticaExpression Vector pZUFmEaD8S, Comprising a Synthetic Δ8 Desaturase Gene(Derived From Euglena anabaena), Codon-Optimized for Expression inYarrowia lipolytica (EaD8S)

The present Example describes the functional expression of Yarrowialipolytica vector pZUFmEaD8S, comprising a chimeric FBAINm::EaD8S::Pex20gene, wherein EaD8S is the synthetic Δ8 desaturase derived from Euglenaanabaena and codon-optimized for expression in Yarrowia (Example 6). Theplasmid pZUFmEaD8S (FIG. 8B) contained the following components:

TABLE 10 Components Of Plasmid pZUFmEaD8S (SEQ ID NO: 51) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:51 Components Swa I/BsiW I FBAINm::EaD8S::Pex20, comprising: (7333-1584)FBAINm: Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356)EaD8S: codon-optimized Δ8 desaturase (SEQ ID NO: 39), derived fromEuglena anabaena Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene(GenBank Accession No. AF054613) 2620-1740 ColE1 plasmid origin ofreplication 3550-2690 Ampicillin-resistance gene (Amp^(R)) for selectionin E. coli 4449-5753 Yarrowia autonomous replication sequence (ARS18;GenBank Accession No. A17608) 7297-5796 Yarrowia Ura 3 gene (GenBankAccession No. AJ306421)Functional Analysis of Yarrowia lipolytica Transformants ComprisingpZUFmEaD8S

Plasmid pZUFmEaD8S (SEQ ID NO:51; FIG. 8B) was transformed into strainY4001U as described in the General Methods. The transformants wereselected on MMLeu plates. After 2 days growth at 30° C., transformantswere picked and re-streaked onto fresh MMLeu plates. Once grown, thesestrains were individually inoculated into 3 mL liquid MMLeu at 30° C.and shaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that there were about 6.5% DGLA and 9.4% EDA of totallipids produced in all 7 transformants, wherein the conversionefficiency of EDA to DGLA in these 7 strains was determined to be about41%.

1. A microbial host cell comprising an isolated polynucleotidecomprising: (a) a nucleotide sequence encoding a polypeptide having Δ8desaturase activity, wherein the polypeptide has at least 80% amino acididentity, based on the Clustal V method of alignment, when compared toan amino acid sequence selected from the group consisting of: SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; (b) a nucleotidesequence encoding a polypeptide having Δ8 desaturase activity, whereinthe nucleotide sequence has at least 80% sequence identity, based on theBLASTN method of alignment, when compared to a nucleotide sequenceselected from the group consisting of: SEQ ID NO:17, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20 and SEQ ID NO:39; (c) a nucleotide sequenceencoding a polypeptide having Δ8 desaturase activity, wherein thenucleotide sequence hybridizes under stringent conditions to anucleotide sequence selected from the group consisting of: SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:39; or (d) acomplement of the nucleotide sequence of (a), (b) or (c), wherein thecomplement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.
 2. The microbial host cell ofclaim 1 wherein the isolated polynucleotide encodes an amino acidsequence selected from the group consisting of: SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23 and SEQ ID NO:24.
 3. The microbial host cell ofclaim 1 wherein the microbial host cell is selected from the groupconsisting of yeast, algae, bacteria, euglenoids, stramenopiles andfungi.
 4. The microbial host cell of claim 3 wherein the cell is afungus of the genus Mortierella sp.
 5. The microbial host cell of claim3 wherein the cell is a stramenopiles selected from the group consistingof: Thraustochytrium sp., and Schizochytrium sp.
 6. The microbial hostcell of claim 3 wherein the yeast is an oleaginous yeast.
 7. Themicrobial host cell of claim 6 wherein the oleaginous yeast is selectedfrom the group consisting of: Yarrowia, Candida, Rhodotorula,Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
 8. A methodfor the production of dihomo-γ-linoleic acid comprising: a) providing amicrobial host cell comprising: (i) a recombinant nucleotide moleculeencoding a Δ8 desaturase polypeptide having at least 80% amino acididentity, based on the Clustal V method of alignment, when compared to apolypeptide having an amino acid sequence selected from the groupconsisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24; and, (ii) a source of eicosadienoic acid; c) growing themicrobial host cell of step (a) under conditions wherein the nucleicacid fragment encoding the Δ8 desaturase polypeptide is expressed andthe eicosadienoic acid is converted to dihomo-γ-linoleic acid; and, d)optionally recovering the dihomo-γ-linoleic acid of step (b).
 9. Amethod for the production of eicosatetraenoic acid comprising: a)providing a microbial host cell comprising: (i) a recombinant nucleotidemolecule encoding a Δ5 desaturase polypeptide having at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto a polypeptide having an amino acid sequence selected from the groupconsisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24; and, (ii) a source of eicosatrienoic acid; b) growing themicrobial host cell of step (a) under conditions wherein the nucleicacid fragment encoding the Δ8 desaturase polypeptide is expressed andthe eicosatrienoic acid is converted to eicosatetraenoic acid; and, c)optionally recovering the eicosatetraenoic acid of step (b).
 10. Themethod of either of claims 8 or 9 wherein the microbial host cell is aYarrowia sp., comprising a recombinant nucleotide molecule encoding a Δ8desaturase polypeptide as set forth in SEQ ID NO:39 wherein therecombinant nucleotide molecule comprises at least 208 codons which areoptimized for expression in Yarrowia.
 11. A method according to eitherof claims 8 or 9 wherein: a.) the recombinant nucleic acid molecule hasa nucleic acid sequence selected from the group consisting of SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:39; and,b.) the host cell is Yarrowia lipolytica.
 12. An isolated nucleic acidmolecule which encodes a Δ8 desaturase as set forth in SEQ ID NO:39wherein at least 208 codons are codon-optimized for expression inYarrowia sp.